Isolation of a rearranged human immunoglobulin gene from a chimeric mouse and recombinant production of the encoded immunoglobulin

ABSTRACT

The specification relates to a method for producing a chimeric non-human animal, which comprises preparing a microcell containing a foreign chromosome(s) or a fragment(s) thereof and transferring the foreign chromosome(s) or fragment(s) thereof into a pluripotent cell by fusion with the microcell; a chimeric non-human animal which can be produced by the above method and its progeny; tissues and cells derived therefrom; and a method for using the same. Further, a pluripotent cell containing a foreign chromosome(s) or a fragment(s) thereof, a method for producing the same, and a method for using the same are also provided. Moreover, a pluripotent cell in which at least two endogenous genes are disrupted, and a method for producing the same by homologous recombination are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part application of InternationalApplication No. PCT/JP96/02427 with an international filing date of Aug.29, 1996.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to chimeric non-human animals, amethod for producing the same and a method for using the same. Thepresent invention allows chimeric non-human animals to retain a foreigngiant DNA fragment(s) of at least 1 Mb and to express the gene(s) onsuch a fragment(s), which was impossible heretofore. Hence, thefollowing becomes possible by using the method.

[0003] Production of animals which retain and express a full length of agene encoding a biologically active substance, for example, a fulllength of human antibody gene. The biologically active substance, forexample, a human-type antibody is useful as a pharmaceutical product.

[0004] Analysis of functions of human giant genes (e.g.,histocompatibility antigen, dystrophin, etc.) in animals.

[0005] Production of model animals with human dominant hereditarydisease and a disease due to chromosomal aberration.

[0006] The present invention relates to pluripotent cells in whichendogenous genes are disrupted, use of the same, and a method forproducing chimeric non-human animals and use of the animals. If aforeign chromosome or a fragment thereof containing a gene encoding agene product identical with or homologous to the gene product encoded bythe disrupted endogenous gene is transferred into the pluripotent cellof the present invention as a recipient cell so that a desiredfunctional cell or a desired chimeric non-human animal is produced fromthe cell, the transferred gene can be expressed efficiently withoutdifferentiation of the pluripotent cell into a germ cell. Even if a germcell of the non-human animal is affected or the pluripotent cell cannotbe differentiated into a germ cell by the disruption of the endogenousgene or the introduction of a foreign gene, a functional cell, or achimeric non-human animal, a tissue or a cell of the animal can retainand express a foreign giant DNA fragment in excess of the heretoforeunattainable 1 Mb (a million bases) in conditions of a deficiency in theendogenous gene and a decrease in the production of an endogenous geneproduct by producing the desired functional cell or non-human animalfrom the pluripotent cell.

[0007] Techniques of expressing foreign genes in animals, that is,techniques of producing transgenic animals are used not only forobtaining information on the gene's functions in living bodies but alsofor identifying DNA sequences that regulate the expression of the genes(e.g., Magram et al., Nature, 315:338, 1985), for developing modelanimals with human diseases (Yamamura et al., “Manual of model mice withdiseases” published by Nakayama Shoten, 1994), for breeding farm animals(e.g., Muller et al., Experientia, 47:923, 1991) and for producinguseful substances with these animals (e.g., Velander et al., P.N.A.S.,89:12003, 1992). Mice have been used the most frequently as hosts forgene transfer. Since mice have been studied in detail as experimentalanimals and the embryo manipulating techniques for mice have beenestablished, they are the most appropriate kind of mammals for genetransfer.

[0008] Two methods are known for transferring foreign genes into mice.One is by injecting DNA into a pronucleus of a fertilized egg (Gordon etal., P.N.A.S., 77:7380, 1980). The other is by transferring DNA into apluripotent embryonic stem cell (hereinafter referred to as “ES cell”)to produce a chimeric mouse (Takahashi et al., Development, 102:259,1988). In the latter method, the transferred gene is retained only in EScell-contributing cells and tissues of chimeric mice whereas it isretained in all cells and tissues of progenies obtained via EScell-derived germ cells. These techniques have been used to produce alarge number of transgenic mice up to now.

[0009] However, there had been a limit of the size of DNA capable ofbeing transferred and this restricts the application range of thesetechniques. The limit depends on the size of DNA which can be cloned.One of the largest DNA fragments which have ever been transferred is aDNA fragment of about 670 kb cloned into a yeast artificial chromosome(YAC) (Jakobovits et al., Nature, 362:255, 1993). Recently, introductionof YAC containing an about 1 Mb DNA fragment containing about 80 percentof variable regions and portions of constant regions (Cμ, Cδ and Cγ) ofa human antibody heavy-chain was reported (Mendes et al., NatureGenetics, 15:146, 1997). These experiments were carried out by fusing aYAC-retaining yeast cell with a mouse ES cell. Although it is believedthat foreign DNA of up to about 2 Mb can be cloned on YAC (Den Dunnen etal., Hum. Mol. Genet., 1:19, 1992), the recombination between homologousDNA sequences occurs frequently in budding yeast cells and therefore, insome cases, a human DNA fragment containing a large number of repeatedsequences is difficult to retain in a complete form. In fact, certainrecombinations occur in 20-40% of the clones of YAC libraries containinghuman genomic DNA (Green et al., Genomics, 11:584, 1991).

[0010] In another method that was attempted, a metaphase chromosome froma cultured human cell was dissected under observation with a microscopeand the fragment (presumably having a length of at least 10 Mb) wasinjected into a mouse fertilized egg (Richa et al., Science, 245:175,1989). In the resulting mice, a human specific DNA sequence (Alusequence) was detected but the expression of human gene was notconfirmed. In addition, the procedure used in this method to preparechromosomes causes unavoidable fragmentation of DNA into small fragmentsdue to the use of acetic acid and methanol in fixing the chromosome onslide glass and the possibility that the injected DNA exists as anintact sequence is small.

[0011] In any event, no case has been reported to date that demonstratessuccessful transfer and expression in mice of uninterrupted foreign DNAfragments having a length of at least 1 Mb.

[0012] Useful and interesting human genes which are desirablytransferred into mice, such as genes for antibody (Cook et al., NatureGenetics, 7: 162, 1994), for T cell receptor (Hood et al., Cold SpringHarbor Symposia on Quantitative Biology, Vol. LVIII, 339, 1993), forhistocompatibility antigen (Carrol et al., P.N.A.S, 84:8535, 1987), fordystrophin (Den Dunnen et al., supra) are known to be such that theircoding regions have sizes of at least 1 Mb. Since human-type antibodiesare important as pharmaceutical products, the production of mice whichretain and express full lengths of genes for human immunoglobulin heavychains (˜1.5 Mb, Cook et al., supra), and light chain κ (˜3 Mb, Zachau,Gene, 135:167, 1993), and light chain λ (˜1.5 Mb, Frippiat et al., Hum.Mol. Genet., 4:983, 1995) is desired but this is impossible to achieveby the state-of-the-art technology (Nikkei Biotec, Jul. 5, 1993).

[0013] Many of the causative genes for human dominant hereditary diseaseand chromosomal aberration which causes congenital deformity (Down'ssyndrome, etc.) have not been cloned and only the information on theapproximate location of the genes on chromosome is available. Forexample, when a gene of interest is found to be located on a specific Gband, which is made visible by subjecting a metaphase chromosome toGiemsa staining, the G band has usually a size of at least several Mb to10 Mb. In order to transfer these abnormal phenotypes into mice, it isnecessary to transfer chromosomal fragments of at least several Mb thatsurround the causative genes, but this is also impossible with thepresently available techniques.

[0014] Hence, it is desired to develop a technique by which a foreignDNA longer than the heretofore critical 1 Mb can be transferred into amouse and expressed in it.

[0015] DNA longer than 1 Mb can be transferred into cultured animalcells by the techniques available today. Such transfer is carried outpredominantly by using a chromosome as a mediator. In the case of human,chromosomes have sizes of about 50-300 Mb. Some methods for chromosometransfer into cells have been reported (e.g., McBride et al., P.N.A.S.,70:1258, 1973). Among them, microcell fusion (Koi et al., Jpn. J. CancerRes., 80:413, 1989) is the best method for selective transfer of adesired chromosome. The microcell is a structural body in which one toseveral chromosomes are encapsulated with a nuclear membrane and aplasma membrane. A few chromosomes (in many cases, one chromosome) canbe transferred by inducing a microcell with an agent that inhibits theformation of spindle in a specific kind of cell, separating themicrocell and fusing it with a recipient cell. The resulting librariesof monochromosomal hybrid cells containing only one human chromosomehave been used for mapping known genes and specifying the chromosomes onwhich unknown tumor-suppressor genes and cellular senescence genes exist(e.g., Saxon et al., EMBO J., 5:3461, 1986). In addition, it is possibleto fragment a chromosome by irradiating a microcell with γ-rays and totransfer part of the fragments (Koi et al., Science, 260:361, 1993). Asdescribed above, microcell fusion is considered to be an appropriatemethod for transferring DNA larger than 1 Mb into a cultured animalcell.

[0016] The expectation that a mouse could be generated from a culturedcell turned to a real fact when the ES cell which has stablepluripotency was discovered (Evans et al., Nature, 292:154, 1981).Foreign genes, various mutations and mutations by targeted generecombination could be introduced into the ES cell, making it possibleto perform a wide variety of genetic modifications in mice (e.g.,Mansour et al., Nature, 336:348, 1988). The ES cell can be used toproduce a mouse having a disrupted target gene by gene targetingtechniques. The mouse is mated with a transgenic mouse having a gene ofinterest to produce a mouse that expresses the gene of interestefficiently. For example, a mouse having a disrupted endogenous antibodygene can be mated with a mouse having a human antibody gene transferredto produce a mouse that expresses the human antibody efficiently. Anormal diploid cell has alleles. A transgenic mouse having one allele ofan mouse antibody heavy-chain gene disrupted expresses an increasedlevel of human antibody in its serum. A mouse having both alleles ofmouse antibody heavy-chain gene disrupted expresses a further remarkablyincreased level of human antibody (S. D. Wagner et al., Genomics,35:405-414, 1996).

[0017] Some researchers have developed a technique in which one alleleof a target gene is disrupted, and then the concentration of a selectivedrug is increased, thereby deleting both alleles of the target gene(double knock-out). However, this technique holds the possibility of adecrease in the ability of the target gene-deficient cell todifferentiate into a germ cell because the target gene-deficient cellobtained by the high-concentration-selective-culture method is culturedin vivo for a long period and because the drug-selection pressure issevere (Takatsu•Taki, Experimental Medicine, supplement, Biomanual UPSeries Basic Techniques for Immunological Study, Yodo-sha, 1995). Inanother case, if two kinds of selective drugs are used for doubleknocking-out, for example, if a neomycin-resistant cell is subjected toa double knock-out treatment with hygromycin, the double drug-resistantES cell is rarely differentiated to produce a mutant mouse (Watanabe etal., Tissue Culture 21, 42-45, 1995). ES cells may lose theirdifferentiation and growth capabilities under certain cultureconditions. When a gene targeting procedure is performed twice, ES cellsdo not lose the ability to differentiate into germ cells of a chimericmouse but the second homologous recombination frequency is extremely low(Katsuki et al., Experimental Medicine, Vol. 11, No. 20, special number,1993). Hence, when a target gene-deficient homozygote is produced,particularly when at least two target genes are targeted, a mousedeficient in each target gene is produced and then the produced mice aremated with each other to produce a homozygote mouse deficient in atleast two genes (N. Longberg et al., Nature, 368:856-859, 1994). Ifgenes to be disrupted exist close to each other and if a mouse deficientin at least two genes cannot be obtained by mating, heterozygote micedeficient in the two target genes are produced from ES cells and theyare mated to produce homodeficient mice (J. H. van Ree et al., Hum MolGenet 4:1403-1409, 1995).

[0018] An attempt to differentiate a pluripotent ES cell into afunctional cell in vitro has been made (T. Nakano et al., Science,265:1098-1101, 1994, A. J. Potocnik et al., The EMBO Journal,13:5274-5283, 1994). The cultivation system used in this attempt, forexample, a system in which the differentiation into a mature B cell canbe induced is expected to be used in the identification of unknowngrowth and differentiation factors which will work in development anddifferentiation processes of B cells.

[0019] As long as the transfer of giant DNA is concerned, it has beenbelieved that the size of the aforementioned foreign DNA fragment whichcan be cloned into a YAC vector is the upper limit. The prior arttechnology of chromosome transfer for introducing a longer DNA intocultured cells has never been applied to gene transfer into mice andthis has been believed to be difficult to accomplish (Muramatsu et al.,“Transgenic Biology”, published by Kodansha Scientific, p.143-, 1989).

[0020] The reasons are as follows.

[0021] The transfer of a human chromosome into a mouse ES cell of anormal karyotype as a recipient cell would be a kind of transfer ofchromosomal aberration. Up to now, it has been believed that geneticaberration at chromosomal levels which is large enough to berecognizable with microscopes is generally fatal to the embryogeny inmice (Gropp et al., J. Exp. Zool., 228:253, 1983 and Shinichi Aizawa,“Biotechnology Manual Series 8, Gene Targeting”, published by Yodosha,1995).

[0022] Available human chromosomes are usually derived from finitelyproliferative normal fibroblasts or differentiated somatic cells such ascancer cells and the like. It was believed that if a chromosome derivedfrom such a somatic cell was transferred into an undifferentiated EScell, the transferred chromosome might cause differentiation of the EScell or its senescence (Muller et al., Nature, 311:438, 1984; Sugawara,Science, 247:707, 1990).

[0023] Only few studies have been reported as to whether a somaticcell-derived chromosome introduced into an early embryo can function inthe process of embryonic development as normally as a germ cell-derivedchromosome to ensure the expression of a specific gene in various kindsof tissues and cells. One of the big differences between the twochromosomes is assumed to concern methylation of the chromosomal DNA.The methylation is changed according to differentiation of cells and itsimportant role in the expression of tissue-specific genes has beensuggested (Ceder, Cell, 53:3, 1988). For example, it has been reportedthat if a methylated DNA substrate is introduced into a B cell, themethylated DNA is maintained after replication and suppresses asite-directed recombination reaction which is essential to theactivation of an antibody gene (Hsieh et al., EMBO J., 11:315, 1992). Inaddition, it was reported that higher levels of de novo methylationoccurred in established cell lines than in vivo (Antequera et al., Cell,62:503, 1990). On the basis of the studies reported, it could not beeasily expected that an antibody gene in a human fibroblast or ahuman-mouse hybrid cell which was likely to be methylated at a highlevel would be normally expressed in a mouse B cell.

[0024] It should be noted that there are two related reports ofIllmensee et al. (P.N.A.S., 75:1914, 1978; P.N.A.S., 76:879, 1979). Onereport is about the production of chimeric mice from fused cellsobtained by fusing a human sarcoma cell with a mouse EC cell and theother is about the production of chimeric mice from fused cells obtainedby fusing a rat liver cancer cell with a mouse EC cell. Many questionsabout the results of the experiments in these two reports were pointedout and thus these reports are considered unreliable (Noguchi et al.,“Mouse Teratoma”, published by Rikogakusha, Section 5, 1987). Althoughit has been desired to perform a follow-up as early as possible, as oftoday when 17 years have passed since the publication of these reports,successful reproduction of these experiments has not been reported.Hence, it is believed that foreign chromosomes cannot be retained andthe genes on the chromosomes cannot be expressed in mice by the methoddescribed in these reports.

[0025] Under these circumstances, it has been believed to be difficultto transfer a giant DNA such as a chromosomal fragment and express it inan animal such as mouse. Actually, no study has been made about thisproblem since the Illmensee's reports.

[0026] Therefore, an object of the present invention is to providechimeric non-human animals which retain foreign chromosomes or fragmentsthereof and express genes on the chromosomes or fragments, and theirprogenies, and a method for producing the same.

[0027] It is also an object of the present invention to providepluripotent cells containing foreign chromosomes or fragments thereofand a method for producing the pluripotent cells.

[0028] Another object of the present invention is to provide tissues andcells derived from the chimeric non-human animals and their progenies.

[0029] A further object of the present invention is to providehybridomas prepared by fusing the cells derived from the chimericnon-human animals and their progenies with myeloma cells.

[0030] A still further object of the present invention is to provide amethod for producing a biologically active substance that is anexpression product of the gene on a foreign chromosome or a fragmentthereof by using the chimeric non-human animals or their progenies, ortheir tissues or cells.

[0031] It is also an object of the present invention to providepluripotent cells which can be used as recipient cells into which aforeign chromosome(s) or a fragment(s) thereof is transfered in theproduction of chimeric non-human animals retaining the foreignchromosome(s) or fragment(s) thereof and expressing a gene(s) on theforeign chromosome(s) or fragment(s) thereof.

[0032] A further object of the present invention is to provide a methodfor using the pluripotent cells.

SUMMARY OF THE INVENTION

[0033] As a result of the various studies conducted to achieve the aboveobjects, the inventors succeeded in transferring chromosomes orfragments thereof derived from human normal fibroblast cells into mouseES cells and obtaining clones which were capable of stable retention ofthe chromosomes or fragments. Moreover, they produced from these ESclones those chimeric mice which retained human chromosomes in normaltissues and which expressed several human genes including human antibodyheavy-chain genes. It has become possible to make that animals retainand express giant DNA fragments by the series of these techniques,although this has been impossible by conventional techniques. Moreover,the inventors succeeded in obtaining embryonic stem cells having both ofantibody heavy-chain and light-chain genes knocked out.

[0034] The subject matter of the present invention is as follows:

[0035] 1. A method for producing a chimeric non-human animal, whichcomprises preparing a microcell containing a foreign chromosome(s) or afragment(s) thereof and transferring the foreign chromosome(s) orfragment(s) into a pluripotent cell by fusion with the microcell.

[0036] 2. A method for producing a pluripotent cell containing a foreignchromosome(s) or a fragment(s) thereof, which comprises preparing amicrocell containing a foreign chromosome(s) or a fragment(s) thereofand transferring the foreign chromosome(s) or fragment(s) thereof into apluripotent cell by fusion with the microcell.

[0037] In the method of item 1 or 2, the foreign chromosome(s) orfragment(s) thereof may be larger than 670 kb, further, at least 1 Mb(one million base pairs). The foreign chromosome or fragment thereof maycontain a region encoding an antibody. The microcell containing aforeign chromosome(s) or a fragment(s) thereof may be induced from ahybrid cell prepared by the fusion of a cell from which the foreignchromosome(s) or fragment(s) thereof is(are) derived, with a cell havinga high ability to form a microcell. The microcell containing a foreignchromosome(s) or a fragment(s) thereof may be induced from a cellprepared by a further fusion of the microcell induced from the hybridcell with a cell having a high ability to form a microcell. The cellfrom which the foreign chromosome(s) or fragment(s) thereof is(are)derived may be a human normal diploid cell. The cell having a highability to form a microcell may be a mouse A9 cell. The pluripotent cellcan be selected from embryonal carcinoma cells, embryonic stem cells,embryonic germ cells and mutants thereof. It is preferred that theforeign chromosome or fragment thereof contains a gene of interest andthat the pluripotent cell has a disrupted gene identical with orhomologous to said gene of interest on the foreign chromosome orfragment thereof. It is also preferred that the foreign chromosome orfragment thereof contains at least two genes of interest and that thepluripotent cell has disrupted genes identical with or homologous tosaid genes of interest on the foreign chromosome or fragment thereof. Inthe pluripotent cell, one or both alleles of a gene identical with orhomologous to the gene of interest on the foreign chromosome or fragmentthereof may be disrupted. The gene of interest may be an antibody gene.The antibody gene may be one or more sets of antibody heavy-chain andlight-chain genes. In the method of item 1 or 2, it is preferred thatthe foreign chromosome or fragment thereof contains a gene of interestand that the foreign chromosome or fragment thereof is transferred intoa pluripotent cell having a disrupted gene identical with or homologousto the gene of interest and then, a chimera is produced from thepluripotent cell by using an embryo of a non-human animal in a straindeficient in an endogenous gene identical with or homologous to the geneof interest. The non-human animal in a strain deficient in an endogenousgene identical with or homologous to the gene of interest can beproduced by homologous recombination in gene targeting. Preferably, thechimeric non-human animal retains the foreign chromosome(s) orfragment(s) thereof, expresses the gene(s) on the foreign chromosome(s)or fragment(s) thereof, and can transmit the foreign chromosome(s) orfragment(s) thereof to its progeny. The chimeric non-human animal ispreferably a mammal, more preferably a mouse.

[0038] 3. A pluripotent cell containing a foreign chromosome(s) or afragment(s) thereof.

[0039] In the pluripotent cell, the foreign chromosome(s) or fragment(s)thereof may be larger than 670 kb. In the cell of item 3, the foreignchromosome or fragment thereof may contain a gene of interest and thepluripotent cell has a disrupted gene identical with or homologous tothe gene of interest on the foreign chromosome or a fragment thereof.The foreign chromosome or fragment thereof may contain at least twogenes of interest and the pluripotent cell has disrupted genes identicalwith or homologous to the genes of interest on the foreign chromosome ora fragment thereof. In the pluripotent cell, one or both alleles of agene identical with or homologous to the gene of interest may bedisrupted. The foreign chromosome or fragment thereof may contain anantibody gene. The antibody gene may be one or more sets of antibodyheavy-chain and light-chain genes. The pluripotent cell can be selectedfrom embryonal carcinoma cells, embryonic stem cells, embryonic germcells and mutants thereof.

[0040] 4. A chimeric non-human animal retaining a foreign chromosome(s)or a fragment(s) thereof and expressing a gene(s) on the foreignchromosome(s) or fragment(s) thereof, or its progeny retaining theforeign chromosome(s) or fragment(s) thereof and expressing the gene(s)on the foreign chromosome(s) or fragment(s) thereof.

[0041] In the chimeric non-human animal or its progeny, the foreignchromosome(s) or fragment(s) thereof may be larger than 670 kb. Theforeign chromosome or fragment thereof may contain a gene of interestand the animal may have a disrupted gene identical with or homologous tothe gene of interest. The foreign chromosome or fragment thereof maycontain at least two genes of interest and the animal may have disruptedgenes identical with or homologous to said genes of interest. In thechimeric non-human animal or its progeny, one or both alleles of a geneidentical with or homologous to the gene of interest may be disrupted.The gene of interest may be an antibody gene. The antibody gene may beone or more sets of antibody heavy-chain and light-chain genes.

[0042] 5. A non-human animal which can be produced by mating thechimeric non-human animals or their progenies of item 4, said non-humananimal retaining the foreign chromosome(s) or fragment(s) thereof andexpressing the gene(s) on the foreign chromosome(s) or fragment(s)thereof, or its progeny retaining the foreign chromosome(s) orfragment(s) thereof and expressing the gene(s) on the foreignchromosome(s) or fragment(s) thereof.

[0043] 6. A non-human animal retaining the foreign chromosome(s) orfragment(s) thereof and expressing a gene(s) on the foreignchromosome(s) or fragment(s) thereof, which can be produced by matingthe chimeric non-human animal or its progeny of item 4, or the non-humananimal or its progeny of item 5, with a non-human animal in a straindeficient in said gene(s) or a gene homologous thereto, or its progenyretaining the foreign chromosome(s) or fragment(s) thereof andexpressing the gene(s) on the foreign chromosome(s) or fragment(s)thereof.

[0044] 7. A tissue from the chimeric non-human animal or its progeny ofitem 4 or from the non-human animal or its progeny of item 5 or from thenon-human animal or its progeny of item 6.

[0045] 8. A cell from the chimeric non-human animal or its progeny ofitem 4 or from the non-human animal or its progeny of item 5 or from thenon-human animal or its progeny of item 6.

[0046] The cell may be a B cell, a primary culture cell derived from ananimal tissue or a cell fused with an established cell.

[0047] 9. A hybridoma prepared by the fusion of the B cell with amyeloma cell.

[0048] 10. A method for producing a biologically active substance, whichcomprises expressing the gene(s) on the foreign chromosome(s) orfragment(s) thereof in the chimeric non-human animal or its progeny ofitem 4, the non-human animal or its progeny of item 5 or the non-humananimal or its progeny of item 6, or a tissue or a cell thereof, andrecovering the biologically active substance as an expression product.

[0049] In the method, the cell of the chimeric non-human animal may be aB cell. The B cell may be immortalized by fusion with a myeloma cell.The chimeric non-human animal cell may be fused with a primary culturecell derived from an animal tissue or fused with an established cellline. The biologically active substance may be an antibody. The antibodyis preferably an antibody of a mammal, more preferably a human antibody.

[0050] 11. A biologically active substance which can be produced by themethod of item 10.

[0051] 12. A non-human animal retaining at least one human antibody genelarger than 670 kb and expressing the gene.

[0052] The non-human animal of item 12 preferably retains at least onehuman antibody gene of at least 1 Mb and expresses the gene. The humanantibody gene may be a human heavy-chain gene, a human light-chain κgene, a human light-chain A gene, or a combination thereof. Thenon-human animal of item 12 may be deficient in a non-human animalantibody gene identical with or homologous to the human antibody gene.The deficiency of non-human animal antibody gene may be caused bydisrupting the non-human animal antibody gene by homologousrecombination.

[0053] 13. A hybridoma prepared by the fusion of a spleen cell of thenon-human animal of item 12 with a myeloma cell.

[0054] 14. An antibody produced by the hybridoma of item 13.

[0055] 15. A non-human animal expressing at least one class or subclassof human antibody.

[0056] The non-human animal of item 15 may be deficient in an endogenousantibody gene identical with or homologous to the expressed humanantibody gene. The class or subclass of human antibody may be IgM, IgG,IgE, IgA, IgD or a subclass, or a combination thereof.

[0057] 16. A non-human animal retaining a foreign DNA(s) larger than 670kb and expressing a gene(s) on the foreign DNA(s).

[0058] The non-human animal of item 16 may be deficient in an endogenousgene identical with or homologous to the expressed gene on the foreignDNA. The non-human animal of item 16 may retain a foreign DNA(s) of atleast 1 Mb and express the gene(s) on the foreign DNA(s). The non-humananimal may be deficient in an endogenous gene identical with orhomologous to the expressed gene on the foreign DNA.

[0059] 17. A method for producing a transgenic non-human animal, whichcomprises preparing a microcell containing a foreign chromosome(s) or afragment(s) thereof, transferring the foreign chromosome(s) orfragment(s) into a cultured cell derived from a blastcyst by fusion withthe microcell and transplanting the nucleus of the cultured cell into anenucleated unfertilized egg.

[0060] 18. A pluripotent cell in which at least two endogenous genes aredisrupted.

[0061] In the cell of item 18, each of the endogenous genes may bedisrupted in one or both alleles. The disrupted endogenous genes may beantibody genes. The disrupted antibody genes may be antibody heavy-chainand light-chain genes. The pluripotent cell can be selected fromembryonal carcinoma cells, embryonic stem cells, embryonic germ cellsand mutants thereof.

[0062] 19. A method of producing the cell of item 18 by at least twohomologous recombinations.

[0063] The method of item 19 may comprise the steps of:

[0064] disrupting one allele of the endogenous gene in the pluripotentcell by homologous recombination using a drug-resistant marker gene;

[0065] culturing the pluripotent cell in the presence of the drug toselect drug-resistant cells; and

[0066] screening the selected drug-resistant cells to yield a cell inwhich both alleles of the endogenous gene have been disrupted.

[0067] In the method of item 19, one allele of the endogenous gene inthe pluripotent cell may be disrupted by homologous recombination usinga drug-resistant marker gene and the other allele of the endogenous genemay be disrupted by another homologous recombination using adrug-resistant marker gene. The same drug-resistant marker gene may beused in the two homologous recombinations. Alternatively, differentdrug-resistant marker genes may be used in the two homologousrecombinations.

[0068] Furthermore, the present invention provides a method of using thepluripotent cell as a recipient cell into which a foreign gene(s) or afragment(s) thereof, or a foreign chromosome(s) or a fragment(s) thereofare to be transferred. The foreign gene(s) or fragment(s) thereof may beincorporated in a vector such as a plasmid, a cosmid, YAC or the like.Alternatively, the foreign chromosome(s) or fragment(s) thereof may becontained in a microcell. The foreign chromosome(s) or fragment(s)thereof is preferably, but not limited to, one that contains a gene(s)identical with or homologous to the endogenous gene(s) disrupted in thepluripotent cell. The term “homologous gene” means herein a geneencoding the same kind of protein or a protein having a similar propertyin the same or different species of a given organism.

[0069] Moreover, the present invention provides a method of using thepluripotent cell for producing a chimeric non-human animal.

[0070] The present invention also provides a method of producing apluripotent cell containing a foreign chromosome(s) or a fragment(s)thereof, which comprises the steps of:

[0071] preparing a microcell containing the foreign chromosome(s) orfragment(s) thereof; and

[0072] fusing the microcell with said pluripotent cell having at leasttwo endogenous genes disrupted, whereby said foreign chromosome(s) orfragment(s) thereof is transferred into said pluripotent cell.

[0073] The present invention further provides a method of producing achimeric non-human animal, which comprises the steps of:

[0074] preparing a microcell containing a foreign chromosome(s) or afragment(s) thereof; and

[0075] fusing the microcell with said pluripotent cell having at leasttwo endogenous genes disrupted, whereby said foreign chromosome(s) orfragment(s) thereof is transferred into said pluripotent cell.

[0076] In the aforementioned two methods, the foreign chromosome(s) orfragment(s) thereof may have a length(s) of at least 1 Mb (100 millionbase pairs). The foreign chromosome(s) or a fragment(s) thereof maycontain a region encoding an antibody. The microcell containing theforeign chromosome(s) or fragment(s) thereof may be induced from ahybrid cell prepared by the fusion of a cell containing the foreignchromosome(s) or fragment(s) thereof, with a cell having a high abilityto form a microcell. The microcell containing the foreign chromosome(s)or fragment(s) thereof may be induced from a cell prepared by a furtherfusion of the microcell induced from the hybrid cell, with a cell havinga high ability to form a microcell. The cell containing the foreignchromosome(s) or fragment(s) thereof may be a human normal diploid cell.The cell having a high ability to form a microcell may be a mouse A9cell. In the methods of producing a chimeric non-human animal, a foreignchromosome(s) or a fragment(s) thereof containing gene(s) identical withor homologous to the endogenous gene(s) disrupted in the pluripotentcell may be transferred into the pluripotent cell having the disruptedat least two endogenous genes and then, a chimera of the cell with anembryo of a non-human animal in a strain deficient in a gene(s)identical with or homologous to said endogenous gene(s) may be prepared.The chimeric non-human animal deficient in a gene identical with orhomologous to the endogenous gene disrupted in said pluripotent cell maybe produced by homologous recombination in gene targeting. The chimericnon-human animal may be such that it retains the foreign chromosome(s)or fragment(s) thereof, expresses a gene(s) on the foreign chromosome(s)or fragment(s) thereof, and can transmit the foreign chromosome(s) orfragment(s) thereof to its progeny. The chimeric non-human animal may bea mammal, preferably a mouse.

[0077] The present invention also provides a pluripotent cell containinga foreign chromosome(s) or a fragment(s) thereof, which is obtainable bya method of producing a chimeric non-human animal, which methodcomprises the steps of:

[0078] preparing a microcell containing the foreign chromosome(s) orfragment(s) thereof; and

[0079] fusing the microcell with said pluripotent cell having at leasttwo endogenous genes disrupted, whereby said foreign chromosome(s) orfragment(s) thereof is transferred into said pluripotent cell. Thepresent invention further provides a method of using the cell forproducing a chimeric non-human animal.

[0080] The present invention also provides a chimeric non-human animalretaining a foreign chromosome(s) or a fragment(s) thereof andexpressing the gene(s) on the foreign chromosome(s) or fragment(s)thereof, which is obtainable by one of the aforementioned methods ofproducing a chimeric non-human animal, or its progeny. The presentinvention also provides a non-human animal retaining a foreignchromosome(s) or a fragment(s) thereof and expressing the gene(s) on theforeign chromosome(s) or fragment(s) thereof which is obtainable bymating between the chimeric non-human animals or its progenies, or itsprogeny. The present invention further provides a tissue from theaforementioned chimeric non-human animal or its progeny, or theaforementioned non-human animal or its progeny. The present inventionstill more provides a cell from the aforementioned chimeric non-humananimal or its progeny, or the aforementioned non-human animal or itsprogeny. The cell may be a B cell.

[0081] The present invention also provides a hybridoma prepared by thefusion of the cell from the aforementioned chimeric non-human animal orits progeny, or the aforementioned non-human animal or its progeny witha myeloma cell.

[0082] The present invention provides a non-human animal or its progenyretaining a foreign chromosome(s) or a fragment(s) thereof andexpressing a gene(s) on the foreign chromosome(s) or fragment(s)thereof, which is obtainable by mating said chimeric non-human animal orits progeny or said non-human animal or its progeny retaining theforeign chromosome(s) or fragment(s) thereof and expressing the gene(s)on the foreign chromosome(s) or fragment(s) thereof, with a non-humananimal in a stain deficient in a gene(s) identical with or homologous tosaid gene(s).

[0083] Furthermore, the present invention provides a method of producinga biologically active substance, which comprises expressing a gene(s) ona foreign chromosome(s) or a fragment in the chimeric non-human animalor its progeny, or the non-human animal or its progeny, or a tissue or acell thereof and recovering the biologically active substance as theexpression product. The cell of the chimeric non-human animal or itsprogeny, or the non-human animal or its progeny may be a B cell. The Bcell may be immortalized by fusion with a myeloma cell. The biologicallyactive substance may be an antibody. The antibody may be an antibody ofmammal, preferably a human antibody.

[0084] Moreover, the present invention provides a method of producing abiologically active substance, which comprises expressing a gene(s) on aforeign chromosome(s) or a fragment in a offspring or a tissue and acell thereof, wherein the offspring is produced by mating the chimericnon-human animal or its progeny, or the non-human animal or its progenyretaining the foreign chromosome(s) or fragment(s) thereof with anon-human animal in a strain deficient in a gene identical with orhomologous to said genes, and expressing the gene(s) on the foreignchromosome(s) or fragment(s) thereof, and recovering the biologicallyactive substance as the expression product.

[0085] The present invention also provides a vector containing a foreignchromosomal gene(s) for use in gene transfer into a non-human animal anda non-human animal cell. The foreign chromosome(s) is preferably onefrom human, more preferably a human chromosome #14 fragment. Thenon-human animal is preferably a mouse.

[0086] The term “allele” is used herein.

[0087] The term “homologous gene” means herein a gene encoding the samekind of protein or a protein having a similar property in the same ordifferent species of a given organism.

[0088] According to the present invention, a chimeric non-human animalretaining a foreign chromosome(s) or a fragment(s) thereof andexpressing the gene(s) on the chromosome(s) or fragment(s) is provided.The chimeric non-human animal of the present invention can be used toproduce biologically active substances.

[0089] According to the present invention, a pluripotent cell retaininga foreign chromosome(s) or a fragment(s) thereof and expressing agene(s) on the chromosome(s) or fragment(s) thereof is provided. Thepluripotent cell can be used for treatment of hereditary diseases, forexample, by bone marrow transplantation.

[0090] According to the present invention, a pluripotent cell having atleast two endogenous genes disrupted is provided. The cell of thepresent invention can be used as a recipient cell for transferring aforeign chromosome(s) or a fragment(s) thereof containing a geneidentical with or homologous to the disrupted endogenous genes toproduce a functional cell or a chimeric non-human animal retaining theforeign chromosome(s) or fragment(s) thereof and expressing the gene(s)on the chromosome(s) or fragment(s). A biologically active substance(s)can be produced as a gene product(s) by expressing the gene(s) on thechromosome(s) or fragment(s) thereof in the chimeric non-human animal orits progeny, or a tissue or a cell thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0091]FIG. 1 shows the results of PCR analysis of an A9 cell retaininghuman chromosome #2 (fragment).

[0092]FIG. 2 shows that human chromosome #22 (fragment) is retained inan E14 drug resistant cell (PCR analysis).

[0093]FIG. 3 is a photograph of electrophoresis patterns showing thathuman L1 sequence is retained in a chimeric mouse produced from a humanchromosome #22-transferred ES cell (Southern analysis).

[0094]FIG. 4 is a photograph of electrophoresis patterns showing thepresence of a human chromosome in organs of a human chromosome #22transferred chimeric mouse (PCR analysis).

[0095]FIG. 5 is a photograph of electrophoresis patterns showing theresults of the expression of human genes in a human chromosome #22transferred chimeric mouse (RT-PCR).

[0096]FIG. 6 is a photograph of electrophoresis patterns showing theresults of the expression of human genes in organs of a human chromosome#22 transferred chimeric mouse (RT-PCR).

[0097]FIG. 7 shows that human chromosome #4 (fragment) is retained in anE14 drug resistant cell (PCR analysis).

[0098]FIG. 8 is a photograph of electrophoresis patterns showing thedetection of human L1 sequence in a human chromosome #4-transferred E14cell clone (Southern analysis).

[0099]FIG. 9 is a photograph of electrophoresis patterns showing thathuman L1 sequence is retained in a chimeric mouse produced from a humanchromosome #4-transferred ES cell (Southern analysis).

[0100]FIG. 10 shows that human chromosome #14 (fragment) is retained ina TT2 drug resistant cell (PCR analysis).

[0101]FIG. 11 is a photograph of electrophoresis patterns showing thepresence of a human chromosome in organs of a chimeric mouse producedfrom a human chromosome #14 transferred ES cell (PCR analysis).

[0102]FIG. 12 shows the results of a test on a tail-derived fibroblastcell for resistance to G418.

[0103]FIG. 13 shows the concentration of human antibody IgM in a serumof a human serum albumin (hereinafter referred to as “HSA”)-immunizedchimeric mouse (ELISA).

[0104]FIG. 14 shows the concentration of human antibody IgG in a serumof an HSA-immunized chimeric mouse (ELISA).

[0105]FIG. 15 shows the results of ELISA of hybridoma clone H4B7 capableof producing human IgM.

[0106]FIG. 16 is a photograph of the results of FISH analysis of a mouseES cell clone (TT2 cell clone PG15) retaining partial fragments of humanchromosomes #2 and 14.

[0107]FIG. 17 shows that the antibody titer of anti-HSA human IgG isincreased in a serum of an HSA-immunized chimeric mouse.

[0108]FIG. 18 shows that the antibody titer of anti-HSA human Igκ isincreased in a serum of an HSA-immunized chimeric mouse.

[0109]FIG. 19 is a photograph of electrophoresis patterns showing thedetection of human L1 sequence in a human chromosome #22-transferred TT2cell clone (Southern analysis).

[0110]FIG. 20 shows that the antibody titer of anti-HSA human Igλ isincreased in a serum of an HSA-immunized chimeric mouse.

[0111]FIG. 21 shows that a partial fragment of human chromosome #2 isretained in a progeny of a chimeric mouse into which a partial fragmentof a human chromosome #2 was transferred (PCR analysis).

[0112]FIG. 22 shows the presence of a cell expressing human μ chain onthe cell surface in a spleen of a human chromosome #14-transferredchimeric mouse (flow cytometry analysis).

[0113]FIG. 23 shows the structure of LoxP-pstNEO plasmid DNA.

[0114]FIG. 24 shows the structure of genomic DNA carrying a mouseantibody heavy chain Cμ gene.

[0115]FIG. 25 shows the structure of genomic DNA carrying a mouseantibody light-chain κ gene.

[0116]FIG. 26 shows the structures of a mouse antibody heavy-chaintargeting vector and a probe for Southern blotting, as well as a DNAfragment to be detected in homologous recombinants.

[0117]FIG. 27 shows the structures of a mouse antibody light-chain κtargeting vector and a probe for Southern blotting, as well as a DNAfragment to be detected in homologous recombinants.

[0118]FIG. 28 is a photograph of electrophoresis patterns showing theresults of Southern blot analysis of mouse antibody heavy-chainhomologous recombinants and high concentration G418 resistant clonesderived therefrom.

[0119]FIG. 29 shows a photograph of electrophoresis patterns showing theresults of Southern blot analysis of mouse antibody light-chainhomologous recombinants.

[0120]FIG. 30 shows the structure of pLoxP-PGKPuro plasmid DNA.

[0121]FIG. 31 shows a mouse antibody light-chain κ targeting vector, aprobe for use in the southern blot analysis of genomic DNA fromtransformant TT2F cells, and DNA fragments to be detected in homologousrecombinants.

[0122]FIG. 32 shows a photograph of electrophoresis patterns showing theresults of Southern blot analysis of high concentration G418 resistantcell clones derived from mouse antibody light-chain homologousrecombinants.

[0123]FIG. 33 shows that the antibody titers of anti-HSA human IgHantibodies are increased in a serum of an HSA-immunized chimeric mouse.

[0124]FIG. 34 is a photograph of the result of FISH analysis of anantibody heavy- and light-chains deficient mouse ES cell clone retainingpartial fragments of human chromosomes #2 and #14.

[0125]FIG. 35 shows that the antibody titers of anti-HSA human Igantibodies are increased in a serum of an HSA-immunized chimeric mouse.

[0126]FIG. 36 shows a photograph of the result of FISH analysis of amouse A9 cell containing human chromosome #14 (human centromere sequenceprobe).

[0127]FIG. 37 shows a photograph of the result of FISH analysis of amouse A9 cell containing human chromosome #14 (human chromosome-specificprobe).

[0128]FIG. 38 shows the results of a test for stability of humanchromosome fragments (#14: SC20, #2:W23) in a mouse ES cell.

[0129]FIG. 39 shows the results of analysis for stability of humanchromosome #14 fragments in a mouse.

[0130]FIG. 40 shows the results of PCR analysis of a G418 resistanthybrid cells retaining human chromosome #22 (fragment).

[0131]FIG. 41 shows the results of FISH analysis of an A9 cell retainingfragmented human chromosome #22.

[0132]FIG. 42 shows the results of complete human antibody-producingmouse strains established by mating.

[0133]FIG. 43 shows the results of the determination of theconcentration of human antibody κ chain in a serum of a mouse retaininga human chromosome #2 fragment, W23.

[0134]FIG. 44 shows the results of the determination of theconcentration of human antibody κ and λ chains in a serum of a mouse.

[0135]FIG. 45 shows the structure of pBS-TEL/LIFPuro.

[0136]FIG. 46 shows that human chromosome #22 is retained in a DT40 cellclone.

[0137]FIG. 47 shows the identification of homologous recombinant in LIFlocus.

[0138]FIG. 48 shows the fragmentation of human chromosome #22 in aDT40/#22neo cell clone.

[0139]FIG. 49 shows a DT40 cell clone retaining full length orfragmented human #22 chromosome.

BEST MODE FOR CARRYING OUT THE INVENTION

[0140] The present invention will now be described in detail.

[0141] A non-human animal that retains a human chromosome(s) or afragment(s) thereof and which expresses the gene on the chromosome(s) orfragment(s) thereof can be produced by

[0142] (1) preparing a chromosome donor cell which retains a labeledhuman chromosome or a fragment thereof;

[0143] (2) transferring the human chromosome or fragment thereof into anon-human animal pluripotent cell by microcell fusion;

[0144] (3) producing a chimeric non-human animal from the cell; and

[0145] (4) confirming that the human chromosome is retained in thechimeric non-human animal and that a human gene is expressed.

[0146] In this procedure, a mouse is used as a non-human animal thatretains a human chromosome or a fragment thereof and which expresses thegene on the chromosome or fragment thereof (the mouse is hereinafterreferred to as a “human chromosome transferred mouse”).

[0147] The term “human chromosome” means a naturally occurring complexwhich consists of nucleic acids and proteins that are derived from humancells. There are 46 normal human chromosomes of 23 kinds (24 kinds inmale), each of which contains DNAs of about 50-300 Mb in size. In thepresent invention, the human chromosome includes not only partialfragments which can be stably replicated and segregated as independentchromosomes but also fragments that are translocated on mousechromosomes and which are retained stably. The size of the DNA isusually at least 1 Mb and in some cases, it is smaller than 1 Mb. Thefeature of the present invention resides in that a mouse can retain andexpress the foreign gene on a foreign chromosome as a mediator withouttreatments such as cloning in an E. coli or yeast cell, or extraction ofthe DNA from a cell.

[0148] The term “human chromosome transferred mouse” means a mouseretaining a human chromosome(s) or a fragment(s) thereof in all or partof its normal somatic cells. The mouse expresses the gene(s) on a humanchromosome(s) or a fragment(s) thereof in all or part of its normalsomatic cells.

[0149] (1) Preparation of a Chromosome Donor Cell Which Retains aLabeled Human Chromosome or a Fragment Thereof

[0150] A desired chromosome donor cell 1) retains a human chromosome(s)labeled with a marker also available for selection of recipient cells;2) does not contain other human chromosomes; and 3) has a higher abilityto form a microcell.

[0151] Any human-derived cell lines, cancer cells and primary culturecells can be used as materials for providing human chromosomes. Amongthem, normal fibroblast cells are suitable because they have a lowpossibility of abnormality such as deletion and amplification ofchromosomes and can be readily cultured.

[0152] As for 1), human cells can be transformed with vectors thatexpress genes for markers such as drug-resistance (e.g., G418-,puromycin-, hygromycin- or blasticidin-resistance). Promoters operatingefficiently not only in human cells but also in recipient cells such asmouse ES cells are desirably used to regulate the expression of themarker used. For this purpose, herpes simplex virus thymidine kinasepromoter linked with SV 40 enhancer (Katoh et al., Cell Struct. Funct.,12:575, 1987), mouse PGK-1 promoter (Soriano et al., Cell, 64:693, 1991)and the like can be used. A library of human cell transformants in whichthe introduced marker genes have been inserted into 46 human chromosomesof 23 kinds at random can be prepared by transformation throughelectroporation (Ishida et al., “Cell Technology Experiment Manual”,published by Kodansha, 1992) and the like and subsequent selection oftransformants.

[0153] As for 3), since many human normal cells have a very low abilityto form microcells, the whole cell of the transformant may be fused witha cell having a high ability to form microcells such as mouse A9 cell(Oshimura, M., Environ. Health Perspect., 93:57, 1991) so as to providethe transformed cell with an ability to form microcells. It is knownthat in mouse-human hybrid cells, human chromosomes selectivelydisappear. The fused cell selected by the marker can retain stably themarked human chromosome.

[0154] In order to meet the condition of 2), it is desired to obtain amicrocell from the fused cell and fuse it again with a mouse A9 cell. Inthis case, too, most of the cells selected by the marker will meet thethree conditions 1), 2) and 3) above. The marked human chromosomes canbe identified in the finally obtained mouse-human monochromosomal hybridcells by PCR (Polymerase Chain Reaction, Saiki et al., Science, 239:487,1988), Southern blot analysis (Ausubel et al., Current protocols inmolecular biology, John Wiley & Sons, Inc., 1994), FISH analysis(Fluorescence In situ Hybridization, Lawrence et al., Cell, 52: 51,1988) and the like. If the transfer of a specified chromosome isdesired, the above procedures are applied to each of many human celltransformant clones to select a clone in which a chromosome of interestis marked. Alternatively, the above procedures are applied to a mixtureof human cell transformant clones and the identification of humanchromosomes is carried out on a large number of the resultingmouse-human monochromosome hybrid cells.

[0155] In addition, a marker gene can be inserted into a desired site byhomologous recombination of a specific DNA sequence on the chromosomewhich is to be transferred (Thomas et al., Cell, 51:503, 1987).

[0156] A microcell prepared from the mouse-human hybrid cell may beirradiated with γ-rays such that the marked human chromosome isfragmented and transferred into a mouse A9 cell. Even if the microcellis not irradiated with γ-rays, a partially fragmented human chromosomemay be transferred at a certain frequency. In these cases, the resultingmicrocell fused clones retain partial fragments of the marked humanchromosomes. These clones can be used when it is desired to transfer thepartial fragments into recipient cells.

[0157] Human chromosomes to be introduced into ES cells may be modifiedby deletion, translocation, substitution and the like. Specificprocedures for these modifications are as follows:

[0158] 1) In each of the steps of preparing the aforementionedmouse-human hybrid cell, inducing a microcell from the mouse-humanhybrid cell, further fusing the microcell with a mouse A9 cell, inducinga microcell from the further fused cell and fusing the latter microcellwith a mouse ES cell, deletion and/or translocation of human chromosomesmay occasionally occur. Cells retaining such mutated chromosomes areselected under the microscopic observation of chromosomes or by use ofPCR, Southern analysis, or the like. A clone retaining a desired mutantchromosome can be selected from a mouse A9 library retaining varioushuman chomosomes. A clone retaining a desired mutant chromosome can beselected from A9 or ES cell fused with a microcell induced from a mouseA9 cell retaining a certain human chromosome. The frequency offragmentation of chromosomes can be raised by γ-ray irradiation (Koi etal., Science, 260:361, 1993).

[0159] 2) A targeting vector retaining a loxp sequence that isrecognized by Cre enzyme is constructed. A clone into which a loxpsequence has been inserted at a desired site on a chromosome is obtainedby homologous recombination in a cell retaining a human chromosome.Subsequently, Cre enzyme is expressed in the cell of the clone to selecta mutant having chromosomal deletion and/or translocation caused bysite-specific recombination. See WO97/49804 and Smith et al., NatureGenetics, 9:376, 195. As a host into which a targeting vector is to beintroduced, a cell allowing for high-frequency homologous recombinationsuch as DT40 cell (Dieken et al., Nature Genetics, 12:174, 1996) mayalso be used.

[0160] 3) A targeting vector retaining a human telomere sequence isconstructed and the telomere sequence is inserted in the cell at adesired site on a chromosome by homologous recombination in a cellretaining a human chromosome. After a clone into which the telomeresequence has been inserted is obtained, a mutant having deletion causedby the telomere truncation is obtained. See Itzhaki et al., NatureGenet., 2, 283-287, 1992 and Brown et al., P. N. A. S., 93:7125, 1996.As a host into which a targeting vector is to be introduced, a cellallowing for high-frequency homologous recombination such as DT40 cell(Dieken et al., supra) may also be used. Telomere truncation of humanchromosomes in DT40 cell is first disclosed in the present invention.Brown (supra) discloses that a vector was inserted into a repeatsequence on a chromosome. However, no specific site can be targeted.Itzhaki et al. discloses that tumor cells, i.e., 12000 cells of cellline HT1080 into which a telomere sequence was introduced were analyzedand 8 homologous recombinants were obtained. They found that out of the8 cells, only one caused deletion by insertion of the telomere sequence.For some kinds of cells, results were reported that no mutant havingtruncation was obtained by insertion of a telomere sequence into somekinds of cells (Barnett et al., Nucleic Acids Res., 21:27, 1993). Inspite of this report, the inventors believed that it was necessary toincrease the absolute number of homologous recombinants in order toobtain mutants having truncation and made an attempt to perform telomeretruncation using a DT40 cell as a host. As a result, it was surprisinglyfound that truncation occurred in all of the 8 homologous recombinantsobtained.

[0161] As mentioned above, a gene that should not be expressed in ahuman chromosome-transferred mouse can be removed by modification of aintroduced chromosome. If the size of a chromosome to be transferred isshortened by fragmentation, the chromosome fragment to be transferredcan be transmitted to progenies of the chromosome-transferred mice. Inaddition, using chromosome translocation and substitution techniques,genes derived from a plurality of chromosomes can be expressed on thesame chromosome fragment and portions of a plurality of genes on thechromosome fragments can be replaced with different genes. In otherwords, foreign chromosome fragments can be used as vectors fortransferring genes into individual mice and their cells.

[0162] (2) Transfer of the Human Chromosome or Fragment Thereof into aMouse Pluripotent Cell

[0163] It has been reported to date that an embyonic carcinoma cell (ECcell, Hanaoka et al., Differentiation, 48:83, 1991), an embyonic stemcell (ES cell, Evans, Nature, 292:154, 1981) or an embyonic germ cell(EG cell, Matsui et al., Cell, 70:841, 1992) that are derived fromvarious strains of mice contribute to the normal somatic cells in mice,or are capable of the production of chimeric mice, by injection into orcoculturing with a mouse early embryo. ES and EG cells have a very highability in this respect and in many cases, they also contribute to germcells thereby making it possible to produce progenies derived from thecells. EC cells can be obtained predominantly from teratocarcinoma; EScells from the inner cell masses of blastocysts; and EG cells fromprimordial germ cells appearing at the early stage of embryogeny. Thesecell lines and their mutants, and any undifferentiated cells that arecapable of differentiation into all or part of the normal somatic cellsin mice can be used as recipient cells for the transfer of humanchromosome in the present invention. In these recipient cells, for thepurpose of achieving advantageous expression of a human gene to beintroduced, a gene or genes such as a mouse gene homologous to the humangene can be disrupted in a chimeric mouse or a chimeric-mouse derivedtissue or cell by using homologous recombination in gene targeting(Joyner et al., Gene Targeting, 1993, IRL PRESS) or other techniques.

[0164] The microcells prepared from the human chromosome donor cells orthe microcells irradiated with γ-rays can be used as materials for thetransfer of human chromosomes into the recipient cells. The humanchromosome can be transferred into the recipient cell through fusion ofthe recipient cell with the microcell by the method described inMotoyuki Shimizu, “Cell Technology Handbook”, published by Yodosha,1992. The microcell donor cells retain markers by which humanchromosomes or fragments thereof can be selected in the recipient cells.The clone containing a gene, a chromosome or a fragment of interest canbe selected by PCR, Southern blot analysis, FISH method or the like inthe same manner as in (1), thus all kinds of human chromosomes orfragments thereof can be transferred. Moreover, if several chromosomesor fragments thereof which contain different selection markers aretransferred sequentially, a recipient cell retaining these chromosomesor fragments at the same time can be obtained. In addition, cloneshaving an increased number of the transferred chromosome can be selectedfrom the clones into which the human chromosome has been transferred.Such selection can be accomplished by increasing the concentration of aselection drug to be added to a culture medium.

[0165] In order to determine whether the recipient cell selected by themarker (e.g., G418 resistance) on the human chromosome retains the wholeor part of the chromosome retained by the donor cell, the followingconfirmative techniques may be employed: Southern blot analysis usingthe genomic DNA extracted from the selected recipient cell, with a humanspecific repeated sequence (L1, Alu, etc.: Korenberg et al., Cell,53:391, 1988) or a human gene used as a probe; and chromosome analysissuch as PCR method using a human gene specific primer or FISH methodusing a human chromosome specific probe (Lichter et al., Human Genetics,80:224, 1988).

[0166] (3) Production of a Chimeric Mouse from the Human ChromosomeTransferred ES Cell

[0167] The method described in Shinichi Aizawa, “Biotechnology ManualSeries 8, Gene Targeting”, published by Yodosha, 1995 may be used toproduce chimeric mice from the ES cell clone obtained in (2). Inselecting factors for efficient production of chimeric mice, such as thedevelopmental stage of the host embryo and its strain, it is desired toemploy the conditions already reviewed for the respective ES cellclones. For example, 8 cell stage embryos derived from Balb/c (albino,CREA JAPAN, INC.) or ICR (albino, CREA JAPAN, INC.) are desirably usedfor CBA×C57BL/6 F1-derived TT2 cell (agouti, Yagi et al., AnalyticalBiochemistry, 214:70, 1993).

[0168] (4) Confirmation of the Retention of the Human Chromosome in theChimeric Mice and the Expression of a Human Gene

[0169] The contribution of the ES cells in mice produced from theembryos into which ES cells were injected can be roughly judged by thecolor of their coat. However, it should be noted that the total absenceof contribution to the coat color does not always lead to the conclusionthat there is no contribution to other tissues. The detailed informationon the retention of the human chromosome in various tissues of thechimeric mice can be obtained by Southern blot analysis using thegenomic DNA extracted from various tissues, by PCR or the like.

[0170] The expression of the gene on the transferred human chromosomecan be confirmed by the following methods. The expression of mRNAtranscribed from the human chromosome can be detected by RT-PCR methodor northern blotting (Ausubel et al., supra) using RNAs derived fromvarious tissues (Kawasaki et al., P.N.A.S., 85:5698, 1988). Theexpression at the protein level can be detected by enzyme immunoassayusing an anti-human protein antibody that is rendered minimal in itsability to enter into a cross reaction with mouse homologous proteins(ELISA, Toyama and Ando, “Monoclonal Antibody Experiment Manual”,published by Kodansha Scientific, 1987; Ishikawa, “Enzyme immunoassaywith Superhigh Sensitivity”, published by Gakkai Shuppan Center, 1993),western blotting (Ausuel et al., supra), isozyme analysis utilizing thedifference in electrophoretic mobility (Koi et al., Jpn. J. Cancer Res.,80:413, 1989) or the like. The retention of the human chromosome in thechimeric mice and the expression of the gene on the human chromosome canbe confirmed by the appearance of the cells expressing a drug resistancemarker gene in primary culture cells derived from the chimeric mice.

[0171] For example, human IgM, IgG, IgA and the like in sera of thechimeric mice which are produced from ES cells retaining humanchromosome #14 on which a gene for human immunoglobulin heavy chainexists can be detected by enzyme immunoassay using an anti-human Igantibody that is rendered minimal in its ability to enter into crossreaction with mouse antibody. Hybridomas capable of producing a humanimmonoglobulin heavy chain can be obtained by ELISA screening ofhybridomas prepared by immunizing the chimeric mouse with ahuman-derived antigen (e.g., HSA) and fusing the spleen cells of theimmunized mice with mouse myeloma cells (Toyama and Ando, “MonoclonalAntibody Experiment Manual”, published by Kodansha Scientific, 1987).

[0172] The method for producing a chimeric non-human animal of thepresent invention has been explained above with reference to the case ofa mouse retaining a human chromosome(s) or a fragment(s) thereof andexpressing the gene(s) on the chromosome(s) or fragment(s). In thepresent invention, chromosomes or fragments thereof to be transferredinto chimeric non-human animals are not limited to those derived fromhumans but include any foreign chromosomes and fragments thereof. Theterm “foreign chromosome” means a chromosome which is transferred into apluripotent cell and, subsequently, the gene on which (or a fragmentthereof) is expressed in a chimeric non-human animal. The organismspecies from which the foreign chromosome is derived is not particularlylimited. Other kinds of chimeric animals such as chimeric rat and pigcan be produced by the method of the present invention. ES cells orES-like cells derived from animals other than mouse were establishedwith rat (Iannaccone et al., Dev. Biol., 163, 288-, 1994), pig (Wheeleret al., Reprod. Fertil. Dev., 6, 563-, 1994) and bovine (Sims et al.,Proc. Natl. Acad. Sci. USA, 91, 6143-6147, 1994) and attempts have beenmade on cyprinodont, chicken and the like (“Transgenic Animal”,Protein•Nucleic Acid•Enzyme, October, 1995, Special Issue, published byKyoritsu Shuppan). It is known that sheep is developed normally from anunfertilized egg transplanted with the nucleus from ES-like cell (EDcell) or epithelial-like cell obtained by subcultivation of the ES-likecell through at least 10 generations (Campbell et al., Nature, 380, 64-,1996). These ES cells and ES-like cells can be used as recipient cellsin the transfer of foreign chromosomes to produce chimeric non-humananimals retaining the foreign chromosomes or fragments thereof andexpressing the genes on the chromosomes or fragments thereof in the samemanner as in the case of mouse.

[0173] In the present invention, pluripotent cells into which a foreignchromosome(s) or a fragment(s) thereof are transferred are not limitedto the ES cells, EC cells and EG cells mentioned above. For example, itis possible to transfer a foreign chromosome(s) or a fragment(s) thereofinto bone marrow stem cells. If these bone marrow stem cells aretransplanted into a living organism, hereditary diseases, etc. may betreated.

[0174] If an ES cell retaining a foreign chromosome(s) or a fragment(s)thereof is differentiated to a germ cell in the chimeric non-humananimal, reproduced progenies will retain the transferred chromosome(s)or fragment(s) thereof and express the gene(s) on the chromosome(s) orfragment(s) thereof.

[0175] The chimeric non-human animals or their progenies can be used toexpress the gene on the foreign chromosome or fragment thereof and torecover the expression product, thereby producing a biologically activesubstance. More specifically, the chimeric non-human animals or theirprogenies can be bred under the conditions for expressing the gene onthe foreign chromosome or fragment thereof to recover the expressionproduct from the blood, ascites and the like of the animals.Alternatively, the tissues or cells of the chimeric non-human animal, orimmortalized cells derived therefrom (e.g., hybridomas immortalized byfusion with myeloma cells) can be cultured under the conditions forexpressing the gene on the foreign chromosome or fragment thereof andthe expression product is thereafter recovered from the culture.Furthermore, a foreign chromosome(s) or a fragment(s) thereof which wasextracted from tissues or cells of these chimeric non-human animals ortheir progenies, or from immortalized cells derived therefrom; the DNAwhich is a component of said foreign chromosome(s) or fragment(s)thereof; or cDNA derived from the foreign chromosome(s) or fragment(s)thereof retained in tissues or cells of the chimeric non-human animalsor their progenies, or in immortalized cells derived therefrom may beused to transform animal cells or insect cells (e.g., CHO cells, BHKcells, hepatoma cells, myeloma cells, SF9 cells) and the transformedcells may be cultured under the conditions for expressing the gene onthe foreign chromosome(s) or fragment(s) thereof to recover theexpression product (e.g., an antibody protein specific to a particularantigen) from the culture. The expression product can be collected byknown techniques such as centrifugation and purified by known techniquessuch as ammonium sulfate fractionation, partition chromatography, gelfiltration chromatography, adsorption chromatography, preparativethin-layer chromatography and the like. The biologically activesubstance includes any kinds of substances encoded on foreignchromosomes, for example, antibodies, particularly human antibodies. Forexample, the human antibody gene on the foreign chromosome can be clonedfrom spleen cells of the chimeric non-human animal or immortalized cellssuch as hybridomas derived therefrom and transferred into Chinesehamster ovary cells (CHO), myeloma cells or the like to produce a humanantibody (Lynette et al., Biotechnology, 10:1121-, 1992; Bebbington etal., Biotechnology, 10:169-, 1992).

[0176] The chimeric mice or their progenies that retain humanchromosomes #2, 14 and/or 22 (or fragments thereof) which can beproduced by the method of the present invention can retain the greaterpart of the functional sequences of respective genes for human antibodyheavy chain on chromosome #14, light chainλ on chromosome #2 and lightchain λ on chromosome #22. Hence, they can produce a wide repertory ofantibodies which are more similar to human antibody repertory, comparedwith known transgenic mice into which parts of human antibody gene havebeen transferred by using yeast artificial chromosomes and the like(Green et al., Nature Genetics, 7, 13-, 1994; Lonberg et al., Nature,368, 856-, 1994). Also, the chimeric mice and their progenies retainingtwo human chromosomes (or fragments) of #2+#14, #22+#14 or othercombination and the mice and their progenies retaining three humanchromosomes (or fragments) of #2+#14+#22 or other combination which areobtainable by mating said chimeric mice and their progenies retainingtwo human chromosomes (or fragments), as produced by the method of theinvention, can produce complete human antibodies both heavy- andlight-chains of which are derived from human. These mice can recognizehuman-derived antigens as foreign substances to cause an immunoreactionwith the antigens, thereby producing antigen-specific human antibodies.These properties can be utilized to produce human monoclonal andpolyclonal antibodies for therapeutic treatments (Green et al, supra;Longberg et al., supra). On the other hand, in order to obtain a humanantibody having high affinity for a particular antigen more efficiently,it is desirable to produce a mouse which produces a human antibody butnot a mouse antibody (Green et al., supra; Lonberg et al., supra). Inthe present invention, this is achieved typically by the followingMethod A or B using known techniques.

[0177] Method A: a method using a mouse antibody-deficient ES cell and amouse antibody-deficient host embryo for chimera production.

[0178] Method B: a method in which a progeny retaining a humanchromosome is obtained from a human chromosome-transferred chimericmouse, followed by mating said progeny with a mouse in a straindeficient in a mouse antibody gene.

[0179] A typical example for each of Methods A and B will be describedbelow specifically.

[0180] Specific Procedures for Method A

[0181] 1. One allele of a mouse antibody heavy-chain gene present in twocopies in a mouse ES cell is disrupted by homologous recombination ingene targeting (Joyner et al., “Gene Targeting”, published by IRL PRESS,1993). A marker gene, such as a G 418 resistance gene, sandwiched withtwo copies of a sequence which can be removed later by site-specificrecombination [for example, lo×P sequence (see recombination with Crerecombinase in Sauer et al., supra; and see also the use of FLPrecombinase-FRT sequence in O'Gorman, Science, 251;1351-, 1991)] isinserted at the site where the targeted gene is disrupted.

[0182] 2. The resultant drug-resistant mouse ES cells in which oneallele of an antibody heavy-chain gene was disrupted is cultured in thepresence of the drug at a high concentration. Then, those clones whichbecame high concentration drug-resistant are selected. By screeningthese clones, clones in which both antibody heavy-chain genes weredisrupted can be obtained (Shinichi Aizawa, supra).

[0183] Alternatively, the other allele of a target gene in thedrug-resistant mouse ES cell in which one allele of the antibodyheavy-chain gene has been disrupted is also disrupted by homologousrecombination. The same procedure may be repeated using a marker geneother than the precedingly inserted marker gene. For example, homologousrecombination is performed using a G418-resistance gene, followed byanother homologous recombination using a puromycin-resistance gene toobtain clones in which both alleles of the antibody heavy-chain genehave been disrupted. When the same marker as the precedingly insertedmarker is used, an enzyme gene that can cause site-specificrecombination between recombinant sequences inserted at the both ends ofthe drug-resistance gene of item 1 is transiently introduced.Subsequently, drug-sensitive clones are selected that are free of thedrug-resistance gene that has been inserted in the target gene. Then, amarker gene is inserted again by homologous recombination in genetargeting to obtain clones in which both alleles of the target gene havebeen disrupted (Seishi Takatsu et al., Experimental Medicine,supplement, Basic Techniques for Immunological Study, p. 255-, 1995,Yodosha).

[0184] 3. An enzyme gene (e.g., a Cre recombinase gene (Sauer et al.,supra)) which causes a site-specific recombination between therecombination sequences inserted at both the ends of the drug-resistancegene in step 1 above is transiently transferred into the mouse ES cellsfrom step 2 above in which both antibody heavy-chain genes weredisrupted. Then, drug-sensitive clones are selected in which thedrug-resistance genes inserted at the sites of both heavy-chain geneswere deleted as a result of recombination between the loxP sequences[Seiji Takatsu et al., “Experimental Medicine (extra number): BasicTechnologies in Immunological Researches”, p. 255-, published byYodosha, 1995].

[0185] 4. The same procedures in steps 1-3 above are repeated for themouse antibody light-chain κ gene to finally obtain drug-sensitiveclones which are completely deficient in antibody heavy-chain andlight-chain κ.

[0186] 5. Human chromosome #14 (fragment) containing a human antibodyheavy-chain gene and marked with a drug-resistance gene (e.g., G418resistance gene) is transferred into the clone from step 4 above(antibody heavy-chain and light-chain κ-deficient mouse ES cell) bymicrocell fusion.

[0187] 6. Human chromosome #2 (fragment) or #22 (fragment) or bothcontaining a human antibody light-chain gene(s) and marked with adrug-resistance gene different from the one used in step 5 above (e.g.,puromycin resistance gene) are transferred into the clone obtained instep 5 above by microcell fusion.

[0188] 7. Chimeric mice are produced from the ES cells obtained in step6 above by using embryos obtained from a mouse in a strain having noability to produce its own antibody (e.g., RAG-2 knockout mouse, Shinkaiet al., Cell, 68:855-, 1992; membrane-type μ chain knockout mouse,Kitamura et al., Nature, 350:423-, 1991) as host embryos.

[0189] 8. Most of the functional B lymphocytes in the resultant chimericmice are derived from the ES cells [Seiji Takatsu et al., “ExperimentalMedicine (extra number): Basic Technologies in ImmunologicalResearches”, p. 234-, published by Yodosha, 1995]. Since those Blymphocytes are deficient in mouse heavy-chain and light-chain κ, theyproduce human antibodies alone mainly as a result of the expression ofthe functional human antibody genes on the transferred chromosomes.

[0190] Specific Procedures for Method B

[0191] 1. Chimeric mice retaining a human chromosome or a fragmentthereof containing human antibody heavy-chain, light-chain κ orlight-chain λ are used to produce a progeny which stably retains thehuman chromosome or fragment thereof and which can transmit it to thenext generation.

[0192] 2. A mouse in a strain which is homozygous regarding thedeficiency in mouse antibody heavy-chain and light-chain K and whichretains human chromosomes containing human antibody heavy-chain(#14)+light-chain κ (#2), heavy-chain (#14)+light-chain λ (#22) orheavy-chain (#14)+light-chain κ (#2)+light-chain λ (#22) is obtained bymating the mouse in a strain expressing human antibody heavy-chain orlight-chain from step 1 above or a mouse in a strain expressing bothhuman antibody heavy and light-chains obtained by mating the mice fromstep 1, with a mouse in a strain deficient in its own antibody genes(e.g., the membrane-type μ chain knockout mouse mentioned above;light-chain κ knockout mouse, Chen et al., EMBO J., 3:821-, 1993). Sincemice in the resultant strain are deficient in mouse antibody heavy-chainand light-chain K genes, they produce human antibodies alone mainly as aresult of the expression of the functional human antibody genes on thetransferred chromosomes.

[0193] Both Method A and Method B may be used not only to yield humanantibodies but also to yield products of any genes located on a foreignchromosome efficiently.

[0194] The present invention will now be explained in greater detailwith reference to the following examples, which do not limit the scopeof the present invention.

EXAMPLE 1

[0195] Production of Chromosome Donor Cell Retaining Human Chromosome(Fragment) Labeled with G418 Resistance

[0196] Plasmid pSTneoB containing a G418 resistance gene (Katoh et al.,Cell Struct. Funct., 12:575, 1987; Japanese Collection of ResearchBiologicals (JCRB), Deposit Number: VE 039) was linearized withrestriction enzyme SailI (TAKARA SHUZO CO., LTD.) and introduced intohuman normal fibroblast cell HFL-1 (obtained from RIKEN Cell Bank,RCB0251). The HFL-1 cells were treated with trypsin and suspended inDulbecco's phosphate-buffered saline (PBS) at a concentration of 5×10⁶cells/ml, followed by electroporation using a Gene Pulser (Bio-RadLaboratories, Inc.) in the presence of 10 μg of DNA (Ishida et al.,“Cell Technology Experiment Procedure Manual”, published by Kodansha,1992). A voltage of 1000 V was applied at a capacitance of 25 μF with anElectroporation Cell of 4 mm in length (165-2088, Bio-Rad Laboratories,Inc.) at room temperature. The electroporated cells were inoculated intoan Eagle's F12 medium (hereinafter referred to as “F12”) supplementedwith 15% fetal bovine serum (FBS) in 3-6 tissue culture plastic plates(Corning) of 100 mmφ. After one day, the medium was replaced with a F12supplemented with 15% FBS and containing 200 μg/ml of G418 (GENENTICIN,Sigma). The colonies formed after 2-3 weeks were collected in 52 groupseach consisting of about 100 colonies. The colonies of each group wereinoculated again into a plate of 100 mmφ and cultured.

[0197] Mouse A9 cells (Oshimura, Environ. Health Perspect., 93:57, 1991;JCRB 0211) were cultured in Dulbecco's modified Eagle's medium(hereinafter referred to as “DMEM”) supplemented with 10% FBS in platesof 100 mmφ. The G418 resistant HFL-1 cells of 52 groups were cultured inF12 supplemented with 15% FBS and 200 μg/ml of G418 in plates of 100mmφ. The mouse A9 cells and HFL-1 cells were treated with trypsin andone fourth to one half of both cells were mixed. The mixed cells wereinoculated into a plate of 100 mmφ and cultured in a mixture of equalamounts of DMEM containing 10% FBS and F12 containing 15% FBS for aperiod ranging from a half day to one day. Cell fusion was carried outin accordance with the method described in Shimizu et al., “CellTechnology Handbook”, published by Yodosha, p.127-, 1992. The cellsurface was washed twice with DMEM and then treated sequentially with 2ml of a PEG (1:1.4) solution for 1 minute and with 2 ml of PEG (1:3) for1 minute. After the PEG solution was sucked up, and the cells werewashed three times with a serum-free DMEM, followed by cultivation inDMEM supplemented with 10% FBS for 1 day. The cells were dispersed bytreatment with trypsin and suspended in a double selective medium (10%FBS supplemented DMEM) containing ouabain (1×10⁻⁵ M, Sigma) and G418(800 μg/ml), followed by inoculation in 3 plates of 100 mmφ. After about3 weeks cultivation, the colonies formed were treated with trypsin todisperse the cells, which were cultured in a selective medium (10% FBSsupplemented DMEM) containing G418 (800 μg/ml).

[0198] The cells were dispersed by treatment with trypsin and two groupsof the cells were collected, followed by cultivation in 6 centrifugeflasks (Coaster, 3025) of 25 cm² until the cell density reached 70-80%confluence. The medium was replaced with a medium (20% FBS supplementedDMEM) containing Colcemid (0.05 μg/ml, Demecolcine, Wako Pure ChemicalsCo., Ltd) and the cells were cultured for 2 days to form microcells.After the culture medium was removed, a cytochalasin B (10 μg/ml, Sigma)solution preliminarily warmed at 37° C. was filled in the 25 cm ²centrifuge flask, which were inserted into an acryl centrifugecontainer, followed by centrifugation at 34° C. at 8,000 rpm for 1 hour.The microcells were suspended in a serum-free medium and purified bypassage through a filter. To the mouse A9 cells cultured to 80%confluence in the flask of 25 cm², the purified micorcells were addedand the two kinds of cells were fused with a PEG solution. The fusedcells were cultured in a G418 containing selective medium and coloniesformed were isolated. Human chromosomes #2, 4, 14 and 22 retained in therespective clones were identified by the methods described in (1)-(3)below. All other experimental conditions such as operating proceduresand reagents-were in accordance with Shimizu et al., “Cell TechnologyHandbook”, published by Yodosha, p127-.

[0199] (1) PCR Analysis

[0200] The isolated cells were cultured and genomic DNA was extractedfrom the cells with a Puregene DNA Isolation kit (Gentra System Co.).PCR was performed using the genomic DNA as a template with humanchromosome specific primers to select the clones retaining humanchromosome #2, 4, 14 or 22. The PCR amplification was conducted withabout 0.1μg of the genomic DNA as a template, using a thermal cycler(GeneAmp 9600, Perkin-Elmer Corp.) in accordance with the methoddescribed in Innis et al., “PCR Experiment Manual”, published by HBJPublication Office, 1991. Taq polymerase was purchased from Perkin-ElmerCorp. and the reaction was performed in a cycle of 94° C., 5 minutes and35 cycles of denaturing at 94° C., 15 seconds, annealing at 54-57° C.,15 seconds (variable with the primers) and extension at 72° C., 20seconds. The gene on each chromosome (O'Brien, Genetic Maps, 6thedition, Book 5, Cold Spring Harbor Laboratory Press, 1993) andpolymorphic markers (Polymorphic STS Primer Pair, BIOS Laboratories,Inc.; Weissenbach et al., Nature 359:794, 1992; Walter et al., NatureGenetics, 7:22, 1994) were used as primers. The primers for the geneswere prepared on the basis of nucleotide sequences obtained from databases such as GenBank, EMBL and the like. The names of the polymorphicprimers and the sequences of the primers for the genes will be shown forthe respective chromosomes in the following examples (#2, Example 1; #4,Example 6, #14, Example 9; #22, Example 2). The following geneticmarkers and polymorphic makers (Polymorphic STS Primer Pairs: D2S207,D2S177, D2S156 and D2S159, BIOS Laboratories, Inc.) were used toidentify chromosome #2.

[0201] Cκ (immunoglobulin kappa constant): 5′-TGGAAGGTGGATAACGCCCT (SEQID NO: 1), 5′-TCATTCTCCTCCAACATTAGCA (SEQ ID NO: 2)

[0202] FABP1 (fatty acid binding protein-1 liver):5′-GCAATCGGTCTGCCGGAAGA (SEQ ID NO: 3), 5′-TTGGATCACTTTGGACCCAG (SEQ IDNO: 4)

[0203] Vk3-2 (immunoglobulin kappa variable): 5′-CTCTCCTGCAGGGCCAGTCA(SEQ ID NO: 5), 5′-TGCTGATGGTGAGAGTGAACTC (SEQ ID NO: 6)

[0204] Vk1-2 (immunoglobulin kappa variable): 5′-AGTCAGGGCATTAGCAGTGC(SEQ ID NO: 7), 5′-GCTGCTGATGGTGAGAGTGA (SEQ ID NO: 8)

[0205] (2) Fluorescence in situ Hybridization (FISH)

[0206] FISH analysis was conducted with probes specific to humanchromosomes #2, 4, 14 and 22 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.)in accordance with the method described in Matsubara et al., “FISHExperiment Protocol”, published by Shujunsha, 1994.

[0207] For example, at least one clone retaining chromosome #2 wasobtained in 10 groups out of 26 groups (745 clones). Among them, only 5clones were positive to all the used primers specific to chromosome #2.FISH analysis was conducted with these clones. FISH analysis wasconducted with probes specific to human chromosomes #2 (CHROMOSOMEPAINTING SYSTEM, Cambio Ltd.) in accordance with the method described inMatsubara et al., “FISH Experiment Protocol”, published by Shujunsha,1994. In the cells positive to all the primers, an intact form of humanchromosome #2 was observed. In some of the clones positive to part ofthe primers, an independent chromosome smaller than human chromosome #2was observed or a cell having a chromosome in a form fusing withchromosomes other than human chromosome #2 was observed (FIG. 1). InFIG. 1, the names of the clones are shown in the horizontal line and theprimers used in the PCR are shown in the left longitudinal line.  showspositive clones and X shows negative clones. The forms of humanchromosome #2 observed by FISH are shown in the bottom line. Nodescription means no performance of experiment.

[0208] A9 cells retaining human chromosomes #4, 14 and 22 were obtainedby the same procedure.

EXAMPLE 2

[0209] Transfer of Human Chromosome #22 into Mouse ES cells by MicrocellFusion

[0210] The mouse A9 cell clones retaining human chromosome #22(hereinafter referred to as “A9/#22”) from Example 1 were used aschromosome donor cells. Mouse ES cell line E14 (obtained from Martin L.Hooper; Hooper et al., Nature, 326:292, 1987) was used as a chromosomerecipient cell. E14 cells were cultured in accordance with the methoddescribed in Aizawa Shinichi, “Biomanual Series 8, Gene Targeting”,published by Yodosha, 1995 and G418 resistant STO cell line (obtainedfrom Prof. Kondo Hisato, Osaka University) treated with mitomycin C(Sigma) was used as a feeder cell. In the first step, microcells wereprepared from about 10⁸ cells of A9/#22 in accordance with the methodreported by Shimizu et al. “Cell Technology Handbook”, published byYodosha, 1992. The total amount of the resulting microcells weresuspended in 5 ml of DMEM. About 10⁷ cells of E14 were dispersed withtrypsin and washed three times with DMEM and suspended in 5 ml of DMEM.The cells were then mixed with the microcells and the mixture wascentrifuged at 1,250 rpm for 10 minutes to remove the supernatant. Theprecipitate was dispersed by tapping and 0.5 ml of a PEG solution(1:1.4) [5 g of PEG 1000 (Wako Pure Chemicals Co., Ltd.) and 1 ml ofDMSO (Sigma) as dissolved in 6 ml of DMEM) was added. The mixture wasleft to stand at room temperature for 1 minute and 30 seconds and 10 mlof DMEM was added slowly. Immediately thereafter, the resulting mixturewas centrifuged at 1,250 rpm for 10 minutes to remove the supernatant.The precipitate was suspended in 30 ml of a medium for ES cells andinoculated into 3 tissue culture plastic plates (Corning) of 100 mm indiameter into which feeder cells were inoculated. After 24 hours, themedium was replaced with a medium supplemented with 300 μg/ml of G418(GENETICIN, Sigma) and medium replacements were thereafter conducteddaily. Drug resistant colonies appeared in 1 week to 10 days. Thefrequency of appearance was 0-5 per 10⁷ of E14 cells. The colonies werepicked up and grown. The cells were suspended in a storage medium (amedium for ES cells+10% DMSO (Sigma)) at a concentration of 5×10⁶ cellsper ml and stored frozen at −80° C. At the same time, genomic DNA wasprepared from 10⁶-10⁷ cells of each drug resistant clone with a PuregeneDNA Isolation Kit (Gentra System Co.).

[0211] Human chromosome #22 was fragmented by irradiating the microcellswith γ rays (Koi et al., Science, 260:361, 1993). The microcellsobtained from about 10⁸ cells of A9/#22 were suspended in 5 ml of DMEMand irradiated with γ rays of 60 Gy on ice with a Gammacell 40 (CanadianAtomic Energy Public Corporation) at 1.2 Gy/min for 50 minutes. Thefusion of γ ray-irradiated microcells and the selection of drugresistant clones were conducted by the same procedure as in the case ofthe unirradiated microcells. As a result, the frequency of theappearance of the drug resistant clones was 1-7 per 10⁷ of E14 cells.The drug resistant clones were stored frozen and DNA was prepared fromthe clones by the same procedure as in the case of the unirradiatedmicrocells.

[0212] The retention of the transferred chromosomes in the unirradiatedmicrocell-transferred drug resistant clones E14/#22-9 and E14/#22-10,and in the γ ray-irradiated microcell-transferred drug resistant clonesE14/#22-14 and E14/#22-25 was confirmed by the methods described in(1)-(3) below.

[0213] (1) PCR Analysis (FIG. 2)

[0214] The presence of the gene on human chromosome #22 (Genetic Maps,supra) and polymorphic markers (Polymorphic STS Primer Pairs: D22S315,D22S275, D22S278, D22S272 and D22S274, BIOS Laboratories, Inc.; Nature359:794, 1992) was detected by a PCR method using the genomic DNA of thedrug resistant clone as a template. The sequences of oligonucleotideprimers for the genes prepared on the basis of nucleotide sequencesobtained from data bases such as GenBank, EMBL and the like aredescribed below.

[0215] PVALB (parvalbumin): 5′-TGGTGGCTGAAAGCTAAGAA (SEQ ID NO: 9),5′-CCAGAAGAATGGTGTCATTA (SEQ ID NO: 10)

[0216] MB (myoglobin) 5′-TCCAGGTTCTGCAGAGCAAG (SEQ ID NO: 11),5′-TGTAGTTGGAGGCCATGTCC (SEQ ID NO:

[0217] DIA1 (cytochrome b-5 reductase): 5′-CCCCACCCATGATCCAGTAC (SEQ IDNO: 13), 5′-GCCCTCAGAAGACGAAGCAG (SEQ ID NO: 14)

[0218] Igλ (immunoglobulin lambda): 5′-GAGAGTTGCAGAAGGGGTGACT (SEQ IDNO: 15), 5′-GGAGACCACCAAACCCTCCAAA (SEQ ID NO: 16)

[0219] ARSA (arylsulfatase A): 5′-GGCTATGGGGACCTGGGCTG (SEQ ID NO: 17),5′-CAGAGACACAGGCACGTAGAAG (SEQ ID NO: 18)

[0220] PCR amplification (Innis et al., supra) was conducted by usingabout 0.1 μg of the genomic DNA as a template with the above 10 kinds ofthe primers. As a result, amplification products having expected lengthswere detected with all the primers in the case of the two unirradiatedclones and with part of the primers in the case of the γ ray-irradiatedtwo clones. The results are shown in FIG. 2. In FIG. 2, a schematicchromosome map based on the G bands of human chrosome #22 and thelocation of some markers on bands are shown at the left side (O'Brien,GENETIC MAPS, 6th edition, BOOK 5, etc.). The arrangement of the geneticand polymorphic markers shows approximate positional relationships onthe basis of the presently available information (Science, HUMAN GENETICMAP, 1994; Nature Genetics, 7:22, 1994; Nature 359:794, 1992, etc.) andthe order is not necessarily correct. With respect to four kinds of theG418 resistant E14 cell clones, the markers for which the expectedamplification products were detected by PCR are shown by ▪ and themarkers for which the expected amplification products were not detectedare shown by □. The results of the observation by FISH analysis areshown at the bottom side. A9/#22 is a chromosome donor cell.

[0221] (2) Southern Blot Analysis

[0222] Southern blot analysis of about 2μg of the genomic DNA digestedwith restriction enzyme BglII (TAKARA SHUZO CO., LTD.) was conducted byusing human specific repeated sequence L1 (10⁴-10⁵ copies were presentper haploid genome, obtained from RIKEN DNA Bank; Nucleic acidsresearch, 13;7813, 1985; pUK19A derived EcoRI-BamHI fragment of 1.4 kb)as a probe in accordance with the method described in Ausubel et al.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 1994.As a result, a large number of bands hybridized with the human L1sequence were detected in DNA of each drug resistant clone. With respectto the unirradiated 2 clones, their patterns and the quantitative ratioof human chromosomal DNA to mouse genomic DNA which could be presumedfrom the density of the respective bands were the same as those ofA9/#22. The total signal intensity of the bands of the γ-ray irradiatedclones correlated with the degree of the deletion confirmed by the PCRanalysis, as compared with that of A9/#22.

[0223] (3) Fluorescence in situ Hybridization (FISH)

[0224] FISH analysis was conducted with probes specific to humanchromosomes #22 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) in accordancewith the method described in Matsubara et al., “FISH ExperimentProtocol”, published by Shujunsha, 1994. As a result, in almost all ofthe observed metaphase spreads, human chromosome #22 was detected in theform of translocation to the mouse chromosome with respect to E14/#22-9and in the form of an independent chromosome with respect to the threeother clones.

[0225] The results of the above experiments demonstrate that theobtained G418 resistant clones E14/#22-9 and E14/#22-10 retained all ormost part of human chromosome #22 whereas the clones E14/#22-14 andE14/#22-25 retained partial fragments of human chromosome #22.

EXAMPLE 3

[0226] Production of Chimeric Mice from the ES Cells Retaining HumanChromosome #22

[0227] General procedures for obtaining mouse embryos, cultivation,injection of the ES cells into the embryos, transplantation to the uteriof foster mothers were carried out in accordance with the methoddescribed in Aizawa Shinichi, “Biomanual Series 8, Gene Targeting”,published by Yodosha, 1995. The cells in a frozen stock of the G418resistant ES clone E14/#22-9 which was confirmed to retain humanchromosome #22 were thawed, started to culture and injected intoblastcyst-stage embryos obtained by mating a C57BL/6×C3H F1 female mouse(CREA JAPAN, INC.) with a C3H male mouse (CREA JAPAN, INC.); theinjection rate was 10-15 cells per embryo. Two and half days after afoster mother [ICR or MCH(ICR)] mouse (CREA JAPAN, INC) was subjected toa pseudopregnant treatment, about ten of the ES cell-injected embryoswere transplanted to each side of the uterus of the foster mother. Theresults are shown in Table 1. TABLE 1 Production of chimeric mice fromthe ES cells retaining human chromosome #22 (fragments) Number of ES EScell G418 cell-injected Number of Number of Contribution clone/humanresistant blastocyst offspring chimeric to coat color chromosome cloneNo. stage embryos mice mice <−10% 10-30% 30%< E14/#22 9 166 29 16 7 3 6

[0228] As a result of the transplantation of a total 166 of injectedembryos, 29 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of E14 cell-derived pale gray coat color in thehost embryo-derived agouti coat color (dark brown). Out of the 29offsprings, 16 mice were recognized to have partial pale gray coatcolor, indicating the contribution of the E14 cells. The maximumcontribution was about 40% in K22-22.

[0229] These results show that the mouse ES cell clone E14/#22-9retaining human chromosome #22 maintains the ability to produce chimera,that is, the ability to differentiate into normal tissues of mouse.

EXAMPLE 4

[0230] Confirmation of Retention of Human Chromosomal DNA in VariousTissues of the Chimeric Mice Derived from the ES Cells Retaining HumanChromosome #22

[0231] In addition to the determination of coat color in Example 3, theretention of the transferred chromosome was confirmed by PCR analysisusing a template genomic DNA prepared from the tail of the chimericmouse. The tail was obtained from the chimeric mouse at least 3 weeksold in accordance with the method described in Motoya Katsuki,“Development Technology Experiment Manual”, published by KodanshaScientific, 1987. Genomic DNA was extracted from the tail with aPuregene DNA Isolation Kit. Out of the polymorphic primers used inExample 2, PVALB and D22S278 were used, with the extracted genomic DNAas a template, to confirm the amplification products. The analysis wasconducted with 10 of the mice in which the contribution to coat colorwas observed. As a result, the products of amplification with at leasteither of the primers were detected in all the mice.

[0232] Southern blot analysis was conducted in the same manner as inExample 2 by using human L1 sequence as a probe with 2 μg of the genomicDNA derived from the tails of the 6 chimeric mice and one non-chimericmouse. As a result, the presence of a large number of human L1 sequencewas observed in all the chimeric mice and their patterns were similar tothose of E14/#22-9. The quantitative ratio to mouse genome was about 10%at maximum (FIG. 3). In FIG. 3, 2 μg of genomic DNA digested with BglIIwas used in each lane. Human L1 sequence labeled with ³ ²P was used as aprobe and signals were detected with Image Analyzer BAS2000 (Fuji PhotoFilm Co., Ltd.). The lanes represent the genomic DNA derived from thetails of the chimeric mice (K22-6, 7, 8, 9, 10, 11 and 12; 9 is thenon-chimeric mouse) and control DNA (C which is a mixture of E14/#22-9genomic DNA and E14 genomic DNA at a weight ratio of 1:9) as countedfrom the right. The DNA molecular weights are shown at the left side andchimerism in the chimeric mice at the right side (−: 0%, +: <10%, and++: 10-30%).

[0233] With respect to the chimeric mouse (K22-7) having about 5%contribution to coat color, genomic DNA was obtained from the brain,liver, muscle, heart, spleen, thymus, ovary and kidney with an ISOGEN(Nippon Gene Co.). For each tissue, PCR analysis was conducted with MBand D1A1 selected from the primers for the genes used in Example 2. As aresult, both primers gave expected amplification products in all thetissues. The results of PCR analysis using DlA1 primer are shown in FIG.4. The PCR products were electrophoresed on a 2% agarose gel and stainedwith ethidium bromide for detection. The lanes in FIG. 4 represent thefollowing from the left: B, brain; L, liver; SM, skeletal muscle; H,heart; Sp, Spleen; Th, thymus; Ov, ovary; K, kidney; nc, non-chimericmouse tail-derived DNA (negative control); pc, human fibroblast cell(HFL-1) DNA (positive control).

[0234] These results show that E14/#22-9 contributed to various normaltissues in the mouse and that it retained human chromosome #22.

EXAMPLE 5

[0235] Expression of the Human Genes in the Chimeric Mouse Derived fromthe ES Cell Retaining Human Chromosome #22

[0236] The tail of the mouse (K22-7) having about 5% contribution tocoat color was frozen with liquid nitrogen and then disrupted for use asa sample for confirming the expression of the human genes. The samplewas a mixture of tissues such as skin, bones, muscles, blood and thelike. Total RNA was extracted from the sample with an ISOGEN (NipponGene Co.) and used in an RT-PCR method to detect mRNAs of humanmyoglobin (MB) and human cytochrome b5 reductase (DlA1). The RT-PCR wasperformed in accordance with the method described in Innis et al., “PCRExperiment Manual”, published by HBJ Publication Office, 1991. Randamhexamer oligonucleotides (final concentration: 100 pmol, TAKARA SHUZOCO., LTD.) were used as primers for reverse transcription and SuperScript (BRL Co.) as reverse transcriptase. The following primers wereused for amplification using cDNA as a template. MB:5′-TTAAGGGTCACCCAGAGACT, (SEQ ID NO:19) 5′-TGTAGTTGGAGGCCATGTCC (SEQ IDNO:20) DIA1: 5′-CAAAAAGTCCAACCCTATCA, (SEQ ID NO:21)5′-GCCCTCAGAAGACGAAGCAG (SEQ ID NO:22)

[0237] As a result, amplification products specific to mRNAs of bothgenes were detected (FIG. 5). The RT-PCR products were electrophoresedon a 2% agarose gel and stained with ethidium bromide for detection. InFIG. 5, M is a marker (HindIII digested λ DNA+HaeIII digested φ X174DNA,TAKARA SHUZO CO., LTD.); MB, human myoglobin; DlA1, human cytochrome b5reductase; and WT, a wild-type C3H mouse.

[0238] With respect to the same individual (K22-7), total RNA wasextracted from the brain, heart, thymus, liver, spleen, kidney, ovaryand skeletal muscle with an ISOGEN and RT-PCR was performed on eachorgan with the above two primers. As a result, expected products ofamplification with DlA1 were observed in all the organs and those withMB were observed only in the heart and skeletal muscle (FIG. 6).Myoglobin is known to be expressed specifically in muscle cells(Bassel-Duby et al., MCB, 12:5024, 1992). Hence, the above results showthat the gene on the transferred human chromosome can be subjected tothe normal tissue-specific regulation in the mouse. The PCR productswere electrophoresed on a 2% agarose gel and stained with ethidiumbromide for detection. In FIG. 6, the lanes represent the following fromthe left: B, brain; H, heart; Th, thymus; L, liver; Sp, spleen; K,kidney; Ov, ovary; SM, skeletal muscle; and M, marker (supra). The lowerband observed in the results of MB are believed to representnon-specific products.

[0239] These results show that the transferred human chromosome #22 canfunction in normal tissues of the chimeric mice.

EXAMPLE 6

[0240] Transfer of Human Chromosome #4 or Fragments Thereof into ESCells

[0241] The mouse A9 cell clone retaining human chromosome #4(hereinafter referred to as “A9/#4”) from Example 1 was used as achromosome donor cell. Mouse ES cell line E14 (see Example 2) was usedas a chromosome recipient cell. The microcell fusion and the selectionof G418 resistant clones were conducted by the same procedures as inExample 2. The frequency of the appearance of the drug resistant cloneswas 1-2 per 10⁷ of E14 cells. The drug resistant clones were storedfrozen and genomic DNA were prepared by the same procedures as inExample 2. The retention of the transferred human chromosome #4 orfragments thereof in the drug resistant clones E14/#4-4, E14/#4-7 andE14/#4-11 was confirmed by the methods described in (1)-(3) below.

[0242] (1) PCR Analysis (FIG. 7)

[0243] The presence of the gene on human chromosome #4 (O'Brien, GeneticMaps, 6th edition, Book 5, Cold Spring Harbor Laboratory Press, 1993)and polymorphic markers (Polymorphic STS Primer Pairs: D4S395, D4S412,D4S422, D4S413, D4S418, D4S426 and F11, BIOS Laboratories, Inc.; Nature359:794, 1992) was detected by a PCR method. The sequences ofoligonucleotide primers for the genes prepared on the basis ofnucleotide sequences obtained from data bases such as GenBank, EMBL andthe like will be described below. HD (huntington disease):5′-TCGTTCCTGTCGAGGATGAA, (SEQ ID NO:23) 5′-TCACTCCGAAGCTGCCTTTC (SEQ IDNO:24) IL-2 (interleukin-2): 5′ATGTACAGGATGCAACTCCTG, (SEQ ID NO:25)5′-TCATCTGTAAATCCAGCAGT (SEQ ID NO:26) KIT (c-kit):5′GATCCCATCGCAGCTACCGC, (SEQ ID NO:27) 5′-TTCGCCGAGTAGTCGCACGG (SEQ IDNO:28) FABP2 (fatty acid binding protein 2, intestinal),5′-GATGAACTAGTCCAGGTGAGTT, (SEQ ID NO:29) 5′CCTTTTGGCTTCTACTCCTTCA (SEQID NO:30)

[0244] PCR amplification was conducted with the above 11 kinds of theprimers. As a result, the amplification products having expected lengthswere detected with all or part of the primers in all the three clones.In the E14/#4-4 and E14/#4-7 clones, the deletion of partial regions wasobserved. The results are shown in FIG. 7. In FIG. 7, a schematicchromosome map based on the G bands of human chromosome #4 and thelocation of some markers on bands are shown at the left side (seeExample 2). The arrangement of the genetic and polymorphic markers showsapproximate positional relationships on the basis of the presentlyavailable information (see Example 2) and the order is not necessarilycorrect. With respect to the three kinds of the G418 resistant E14 cellclones, the markers for which the expected amplification products weredetected are shown by ▪ and the markers for which the expectedamplification products were not detected are shown by □. The results ofthe observation by FISH analysis are shown at the lower side. A9/#4 is achromosome donor cell.

[0245] (2) Southern Blot Analysis (FIG. 8)

[0246] Southern blot analysis was conducted by the same procedure as inExample 2 using human L1 sequence as a probe with genomic DNAs obtainedfrom E14/#4-4 and E14/#4-7. As a result, a large number of bandshybridized with the human L1 sequence were detected in DNAs of both drugresistant clones. The total signal intensity correlated with the degreeof the deletion confirmed by the PCR analysis, as compared with that ofA9/#4. In FIG. 8, 2 μg of genomic DNA digested with BglII was used ineach lane. Human L1 sequence labeled with ³ ²P was used as a probe andthe signals were detected with an Image Analyzer (BAS 2000, Fuji PhotoFilm Co., Ltd.). In FIG. 8, the lanes represent the following as countedfrom the left: 1, A9/#4 (chromosome donor cell); 2, A9/#4+A9 (1:2); 3,A9/#4+A9 (1:9); 4, A9; 5, E14/#4-7; and 6, E14/#4-4. Lanes 2 and 3represent mixtures of two kinds of DNAs at the ratios shown inparentheses. The molecular weights of DNAs are shown at the left side.

[0247] (3) Fluorescence in situ Hybridization (FISH)

[0248] FISH analysis was conducted with probes specific to humanchromosomes #4 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) by the sameprocedure as in Example 2. As a result, in almost all of the observedmetaphase spreads of the three clones used, human chromosome #4 orpartial fragments thereof were detected in the form of translocation tothe mouse chromosome with respect to E14/#4-4 and in the form of anindependent chromosome with respect to the two other clones. Therelative sizes of the observed human chromosome were consistent withthose presumed from the results of the PCR analysis.

[0249] The results of the above experiments demonstrate that theobtained G418 resistant clones retained the whole human chromosome #4 orpartial fragments thereof.

EXAMPLE 7

[0250] Production of Chimeric Mice from the ES Cells Retaining HumanChromosome #4 Fragments

[0251] The cells in frozen stocks of the G418 resistant ES cell clonesE14/#4-4 and E14/#4-7 which were confirmed to retain partial fragmentsof human chromosome #4 were thawed, started to culture, and injectedinto blastcyst stage embryos obtained by the same method as in Example3; the injection rate was 10-15 cells per embryo. Two and half daysafter a foster mother [ICR or MCH(ICR)] mouse (CREA JAPAN, INC.) wassubjected to a pseudopregnant treatment, about ten of the EScell-injected embryos were transplanted to each side of the uterus ofthe foster mother. The results are shown in Table 2. TABLE 2 Productionof chimeric mice from the E14 cell clones retaining human chromosome #4(fragments) Number of ES ES cell G418 cell-injected Number of Number ofContribution clone/human resistant blastocyst offspring chimeric to coatcolor chromosome clone No. stage embryos mice mice <10% 10-30% 30%<E14/#4 4 160 8 5 5 — — 7 80 5 2 1 1 —

[0252] As a result of the transplantation of a total of 240 injectedembryos, 13 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of E14 cell-derived pale gray coat color in thehost embryo-derived agouti coat color (dark brown). Out of the 13offsprings, 7 mice were recognized to have partial pale gray coat color,indicating the contribution of the E14 cells. The maximum contributionwas about 15% in one individual derived from E14/#4-7.

[0253] These results show that the mouse ES cell clones E14/#4-4 andE14/#4-7 which retain fragments of human chromosome #4 maintain theability to produce chimera, that is, the ability to differentiate intonormal tissues of mouse.

EXAMPLE 8

[0254] Confirmation of Retention of Human Chromosomal DNA in theChimeric Mice Derived from the ES Cells Retaining Partial Fragments ofHuman Chromosome #4 and Expression of the G418 Resistance Gene

[0255] (1) PCR Analysis

[0256] Using the chimeric mice produced in Example 7, genomic DNAs wereprepared from the tails of one individual derived from E14/#4-7(K/#4-7-1: about 5% chimerism) and one individual derived from E14/#4-4(K/#4-4-41: about 5% chimerism) by the same procedure as in Example 4.These DNAs were used as templates to conduct PCR analysis usingpolymorphic marker F11 for chromosome #4 analysis (see Example 6) whichwas detected in E14/#4-7 and E14/#4-4. As a result, expectedamplification products were detected in both mice.

[0257] (2) Southern Analysis (FIG. 9)

[0258] Southern analysis was conducted in the same manner as in Example2 by using human L1 sequence as a probe with 2 μg of the genomic DNAderived from the tail of one individual derived from E14/#4-7 (K/#4-7-1:about 5% chimerism). As a result, the presence of a large number ofhuman L1 sequence was observed and their patterns were similar to thoseof E14/#4-7. The quantitative ratio to mouse genome was about 10% ofthat of E14/#4-7 at maximum. In FIG. 9, 2 μg of genomic DNA digestedwith BglII was used in each lane. Human L1 sequence labeled with ³²P wasused as a probe and signals were detected with Image Analyzer BAS2000(Fuji Photo Film Co., Ltd.). The molecular weights of DNAs are shown atthe left side. The lanes represent the following as counted from theleft: 1, K/#4-7-1; 2, blank; and 3, E14/#4-7.

[0259] (3) Test on the Tail-Derived Fibroblast Cells for G418 Resistance

[0260] Fibroblast cells were prepared from the tails of one individualderived from E14/#4-7 (K/#4-7-1: about 5% chimerism) and one individualderived from E14/#4-4 (K/#4-4-41: about 5% chimerism). In the sameprocedure as in Example 4, the tail of each mouse was cut at a length of5-10 mm and washed several times with PBS/1 mM EDTA, followed bynotching of the tail with a knife. The outer skin layer was removed andthe inner tissues were cut into fine pieces. The fine pieces of tissueswere transferred into a tube containing 5 ml of PBS/1 mM EDTA and leftto stand for 30 minutes to 1 hour at room temperature. Subsequently, thesupernatant was removed leaving a 1 ml portion of the PBS/EDTA behind,and 1 ml of 0.25% trypsin/PBS was added. The tissues were dispersedthoroughly by tapping or pipetting at room temperature for 5-10 minutes.After centrifugation at 1,000 rpm for 10 minutes, the precipitate wassuspended in 2 ml of DMEM (10% FCS) and inoculated into a 35 mm plate.After cultivation for 7-10 days, the cells were treated with trypsin andabout 10⁴ cells per plate were inoculated into two 35 mm plates. G418was added to the medium in one plate at a final concentration of 400μg/ml. The cells were cultured for 5-7 days and the appearance of viablecells in each plate were examined. Under these conditions, 100% of thewild-type ICR mouse-derived fibroblast cells were killed in the presenceof G418. As a result, G418 resistant fibroblast cells was present inboth mice.

[0261] These results show that E14/#4-7 and E14/#4-4 contributed tovarious normal tissues in the mouse and that they retained partialfragments of human chromosome #4.

EXAMPLE 9

[0262] Transfer of Human Chromosome #14 or Fragments thereof into MouseES Cells

[0263] The mouse A9 cell clone retaining human chromosome #14(hereinafter referred to as “A9/#14”) from Example 1 was used as achromosome donor cell. Mouse ES cell line TT2 (purchased from LifetechOriental Co., Yagi et al., Analytical Biochem., 214:70, 1993) was usedas a chromosome recipient cell. The TT2 cells were cultured inaccordance with the method described in Aizawa Shinichi, “BiomanualSeries 8, Gene Targeting”, published by Yodosha, 1995 and G418 resistantprimary culture cells (purchased from Lifetech Oriental Co.) treatedwith mitomycin C (Sigma) were used as feeder cells. The microcell fusionand the selection of G418 resistant clones were conducted by the sameprocedures as in Example 2. The frequency of the appearance of the drugresistant clones was 3-6 per 10⁷ of TT2 cells. The drug resistant cloneswere stored frozen and genomic DNA was prepared by the same proceduresas in Example 2.

[0264] Human chromosome #14 was fragmented by irradiating the microcellswith γ-rays (Koi et al., Science, 260:361, 1993). The microcellsobtained from about 10⁸ cells of A9/#14 were suspended in 5 ml of DMEMand irradiated with γ-rays of 30 Gy on ice with a Gammacell 40 (CanadianAtomic Energy Public Corporation) at 1.2 Gy/min for 25 minutes. Thefusion of γ ray-irradiated microcells and the selection of drugresistant clones were conducted by the same procedure as in the case ofthe unirradiated micorcells. As a result, the frequency of theappearance of the drug resistant clones was 3 per 10⁷ of TT2 cells. Thedrug resistant clones were frozen stored and DNA was prepared by thesame procedure as in Example 2.

[0265] The retention of human chromosome #14 or partial fragmentsthereof in the unirradiated microcell-transferred G418 resistant clones1-4 and 1-5, and in the G418 resistant clones 3-1 and 3-2 (a total of 4clones) into which the γ-ray-irradiated microcell was transferred wasconfirmed by the methods described in (1) and (2) below.

[0266] (1) PCR Analysis (FIG. 10)

[0267] The presence of the gene on human chromosome #14 (O'Brien,Genetic Maps, 6th edition, Book 5, Cold Spring Harbor Laboratory Press,1993) and polymorphic markers (Polymorphic STS Primer Pairs: D14S43,D14S51, D14S62, D14S65, D14S66, D14S67, D14S72, D14S75, D14S78, D14S81,and PCI, BIOS Laboratories, Inc.; Nature 359:794, 1992; Nature Genetics,7:22, 1994) was detected by a PCR method using genomic DNA of the drugresistant clone as a template. The sequences of oligonucleotide primersfor the genes prepared on the basis of nucleotide sequences obtainedfrom data bases such as GenBank, EMBL and the like are described below.NP (nucleoside phosphorylase): 5′-ATAGAGGGTACCCACTCTGG, (SEQ ID NO:31)5′-AACCAGGTAGGTTGATATGG (SEQ ID NO:32) TCRA (T-cell receptor alpha):5′-AAGTTCCTGTGATGTCAAGC, (SEQ ID NO:33) 5′-TCATGAGCAGATTAAACCCG (SEQ IDNO:34) MYH6 (myosin heavy chain cardiac): 5′-TGTGAAGGAGGACCAGGTGT, (SEQID NO:35) 5′-TGTAGGGGTTGACAGTGACA (SEQ ID NO:36) IGA2 (immunoglobulinalpha-2 constant): 5′-CTGAGAGATGCCTCTGGTGC, (SEQ ID NO:37)5′-GGCGGTTAGTGGGGTCTTCA (SEQ ID NO:38) IGG1 (immunoglobulin gamma-1constant): 5′-GGTGTCGTGGAACTCAGGCG, (SEQ ID NO:39)5′-CTGGTGCAGGACGGTGAGGA (SEQ ID NO:40) IGM (immunoglobulin mu constant):5′-GCATCCTGACCGTGTCCGAA, (SEQ ID NO: 41) 5′-GGGTCAGTAGCAGGTGCCAG (SEQ IDNO:42) IGVH3 (immunoglobulin heavy variable-3): 5′-AGTGAGATAAGCAGTGGATG,(SEQ ID NO:43) 5′-GTTGTGCTACTCCCATCACT (SEQ ID NO:44)

[0268] PCR amplification was conducted using the genomic DNAs of the 4drug resistant clones as templates with the above 18 kinds of theprimers by the same procedure as in Example 2. As a result, expectedamplification products were detected with all or part of the primers. Inthe drug resistant clones 3-1 and 3-2 obtained by using the γ-rayirradiated microcells, a tendency for the deletion of partial regions ofchromosome #14 was observed. In the case where the unirradiatedmicrocells were used, deletion was observed as in the case of the 1-4clone. The results are shown in FIG. 10. In FIG. 10, a schematicchromosome map based on the G bands of human chromosome #14 and thelocation of some markers on bands are shown at the left side (seeExample 2). The arrangement of the genetic and polymorphic markers showsapproximate positional relationships on the basis of the presentlyavailable information (see Example 2) and the order is not necessarilycorrect. With respect to four kinds of the G418 resistant TT2 cellclones, the markers for which the expected amplification products weredetected are shown by ▪ and the markers for which the expectedamplification products were not detected are shown by □. A9/#14 is achromosome donor cell. The results of Example 11 (1) are shown at theright side.

[0269] (2) Fluorescence in situ Hybridization (FISH)

[0270] FISH analysis was conducted with probes specific to humanchromosomes #14 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) in accordancewith the method described in Matsubara et al., “FISH ExperimentProtocol”, published by Shujunsha, 1994. As a result, in almost all ofthe observed metaphase spreads of all the 4 clones, human chromosome #14or partial fragments thereof were detected in the form of an independentchromosome. The relative sizes of the observed human chromosome wereconsistent with those presumed from the results of the PCR analysis.

[0271] The results of the above experiments demonstrate that theobtained G418 resistant clones 1-4, 1-5, 3-1 and 3-2 retained the wholeor partial fragments of human chromosome #14.

EXAMPLE 10

[0272] Production of Chimeric Mice from the ES Cells Retaining HumanChromosome #14 or Fragments Thereof

[0273] The cells in the frozen stocks of four G418 resistant ES cellclones (1-4, 3-1, 3-2 and 1-5) that were prepared in Example 9 and whichwere confirmed to retain human chromosome #14 or fragments thereof werethawed, started to culture and injected into 8-cell stage embryosobtained by mating [ICR or MCH(ICR)] male and female mice (CREA JAPAN,INC.); the injection rate was 8-10 cells per embryo. The embryos werecultured in an ES medium overnight to develop to blastocysts. Two andhalf days after a foster mother ICR mouse (CREA JAPAN, INC.) wassubjected to a pseudopregnant treatment, about ten of the injectedembryos were transplanted to each side of the uterus of the fostermother. The results are shown in Table 3. TABLE 3 Production of chimericmice from the TT2 cell clones retaining human chromosome #14 (fragments)Number of ES ES cell G418 cell-injected Number of Number of Contributionclone/human resistant 8-cell offspring chimeric to coat color chromosomeclone No. stage embryos mice mice <20% 20-50% 50-80% TT2/#14 1-4 98 20 1— — 1 1-5 110 14 2 1 — 1 3-1 103 11 2 1 1 — 3-2 183 19 3 — 2 1

[0274] As a result of the transplantation of a total of 494 injectedembryos, 64 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 64produced offsprings, 8 mice were recognized to have partial agouti coatcolor, indicating the contribution of the ES cells. The maximumcontribution was about 80% in one individual derived from 1-4.

[0275] These results show that the G418 resistant ES cell clones (1-4,1-5, 3-1 and 3-2) retaining human chromosome #14 or fragments thereofmaintain the ability to produce chimera, that is, the ability todifferentiate into normal tissues of mouse.

EXAMPLE 11

[0276] Confirmation of Retention of Human Chromosome #14 Fragment DNA inthe Chimeric Mice Derived from the ES Cells Retaining Human Chromosome#14 Fragments

[0277] The retention of human chromosome #14 partial fragments in thechimeric mice obtained in Example 10 was confirmed by the methodsdescribed in (1)-(3) below.

[0278] (1) PCR Analysis using DNAs Derived from Various Tissues

[0279] Genomic DNA was extracted from the tail of one individual derivedfrom 3-1 (K3-1-1: about 25% chimerism) by the same procedure as inExample 4. The DNA was used as a template to conduct PCR analysis usingall of the 14 primers for chromosome #14 analysis which were detected in3-1. As a result, expected amplification products were detected with allthe 14 primers. (FIG. 10)

[0280] With respect to the same individual (K3-1-1), genomic DNA wasobtained from the brain, kidney, spleen, heart, liver and thymus with aPuregene DNA Isolation Kit. For each tissue, PCR analysis was conductedwith IGM primers (see Example 9). As a result, expected amplificationproducts were detected in all the tissues (FIG. 11). The PCR productswere electrophoresed on a 2% agarose gel and stained with ethidiumbromide for detection. In FIG. 11, the lanes represent the following ascounted from the left: B, brain; K, kidney; Sp, Spleen; H, heart; L,liver; Th, thymus; pc, human fibroblast cell (HFL-1) DNA (positivecontrol); nc, non-chimeric mouse tail DNA (negative control); and M,marker (HindIII digested λ DNA+HaeIII digested φ X174 DNA, TAKARA SHUZOCO., LTD.).

[0281] (2) Test on the Tail-Derived Fibroblast Cells for G418 Resistance

[0282] Fibroblast cells were prepared from the tails of two individualsderived from 3-2 (K3-2-1: about 25% chimerism, and K3-2-3: about 50%chimerism) and one individual derived from 1-4 (K1-4-1: about 80%chimerism). In the same procedure as in Example 4, the tail of eachchimeric mouse of 3-6 weeks was cut at a length of 5-10 mm and washedseveral times with PBS/1 mM EDTA, followed by notching of the tail witha knife. The outer layer was removed and the inner tissues were cut intofine pieces. The fine pieces of tissues were transferred into a tubecontaining 5 ml of PBS/1 mM EDTA and left to stand for 30 minutes to 1hour at room temperature. Subsequently, the supernatant was removedleaving a 1 ml portion of the PBS/EDTA behind, and 1 ml of 0.25%trypsin/PBS was added. The tissues were dispersed thoroughly by tappingor pipetting at room temperature for 5-10 minutes. After centrifugationat 1,000 rpm for 10 minutes, the precipitate was suspended in 2 ml ofDMEM (10% FCS) and inoculated into a 35 mm plate. After cultivation for7-10 days, the cells were treated with trypsin and about 10⁴ cells perplate were inoculated into four 35 mm plates. G418 was added to themedium in two of the plates at a final concentration of 400 μg/ml. Thecells were cultured for 5-7 days and the viable cells in each plate werecounted. Under these conditions, 100% of the wild-type ICR mouse-derivedfibroblast cells were killed in the presence of G418. Assuming the samegrowth rate of the G418 resistant fibroblast in the non-selective andselective media, the ratio of the viable cells in the selective mediumto those in the non-selective medium is believed to reflect thecontribution in the fibroblast cell populations of the G418 resistant EScell-derived fiblablast. As a result, the presence of G418 resistantfibroblast cells was observed in all the three individuals as shown inFIG. 12. In FIG. 12, % resistance is an average of 2 pairs of theselective/non-selective 35 mm plates for each mouse. ICR refers to thewild-type ICR mice.

[0283] (3) FISH Analysis of the Tail-Derived G418 Resistant FibroblastCells

[0284] FISH analysis of the K3-2-3 and K1-4-1 derived G418 resistantfibroblast cells obtained in (2) was conducted by the same procedure asin Example 2. Total human DNA extracted from the HFL-1 cells (Example 1)was labeled with FITC so that is could be used as a probe (Matsubara etal., “FISH Experimental Protocol”, published by Shujunsha, 1994). As aresult, in almost all of the observed metaphase spreads of the bothindividuals, partial fragments of the human chromosome in independentforms were observed.

[0285] These results show that the TT2 cell clones retaining fragmentsof human chromosome #14 contributed to various normal tissues in themouse individuals and that they retained partial fragments of humanchromosome #14.

EXAMPLE 12

[0286] Transfer of Partial Fragments of Human Chromosome #2 into ESCells

[0287] The mouse A9 cell W23 retaining a human chromosome #2 fragment(hereinafter referred to as “A9/∩2 W23”) from Example 1 was used as achromosome donor cell. Mouse ES cell line TT2 (see Example 9) was usedas a chromosome recipient cell. The microcell fusion and the selectionof G418 resistant clones were conducted by the same procedures as inExample 2. The frequency of the appearance of the drug resistant cloneswas 1-3 per 10⁷ of TT2 cells. The drug resistant clones were storedfrozen and genomic DNA was prepared by the same procedures as in Example2. The retention of partial fragments of human chromosome #2 in drugresistant clones 5-1, 5-2 and 5-3 was confirmed by the methods describedin (1) and (2) below.

[0288] (1) PCR Analysis

[0289] The presence of Cκ and FABP1 that are the genes on humanchromosome #2 (Genetic Maps, supra) and which were detected in thechromosome donor cell A9/∩2 W23 was detected by a PCR method.

[0290] As a result of PCR amplification using each primer, expectedamplification products were detected with both primers in all of the 3clones.

[0291] (2) Fluorescence in situ Hybridization (FISH)

[0292] FISH analysis was conducted with probes specific to humanchromosome #2 (CHROMOSOME PAINTING SYSTEM, Cambio Ltd.) by the samemethod as in Example 2. As a result, in almost all of the observedmetaphase spreads of the 3 clones, partial fragments of human chromosome#2 in the form of independent chromosomes were detected. The sizes ofthe observed human chromosome were the same as those observed in A9/∩2W23.

[0293] The results of the above experiments demonstrate that theobtained G418 resistant clones retained partial fragments of humanchromosome #2.

EXAMPLE 13

[0294] Production of Chimeric Mice from the ES Cells Retaining HumanChromosome #2

[0295] The cells in a frozen stock of the G418 resistant ES cell clone5-1 that was obtained in Example 12 and which was confirmed to retainhuman chromosome #2 was thawed, started to culture and injected into8-cell stage embryos obtained by mating ICR or MCH(ICR) male and femalemice (CREA JAPAN, INC.); the injection rate was 10-12 cells per embryo.The embryos were cultured in an ES medium (Example 9) overnight todevelop to blastocysts. Two and half days after a foster mother ICRmouse (CREA JAPAN, INC.) was subjected to a pseudopregnant treatment,about ten of the injected embryos were transplanted to each side of theuterus of the foster mother. The results are shown in Table 4. TABLE 4Production of chimeric mice from the TT2 cell clone retaining humanchromosome #2 (fragments) Number of ES ES cell G418 cell-injected Numberof Number of Contribution clone/human resistant 8 cell offspringchimeric to coat color chromosome clone No. stage embryos mice mice <20%20-50% 50-80% TT2/#2 5-1 264 51 18 7 5 6 (W23)

[0296] As a result of the transplantation of a total of 264 injectedembryos, 51 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 51produced offsprings, 18 mice were recognized to have partial agouti coatcolor, indicating the contribution of the ES cells. The maximumcontribution was about 80%.

[0297] These results show that the G418 resistant ES cell clone (5-1)retaining a fragment of human chromosome #2 maintains the ability toproduce chimera, that is, the ability to differentiate into normaltissues of mouse individual.

EXAMPLE 14

[0298] Detection of Human Antibody Heavy Chain in sera of the HumanChromosome #14 Transferred Chimeric Mice

[0299] The concentrations of human antibody in the sera were determinedby enzyme-linked immunosorbent assay (ELISA). The ELISA for humanantibody was performed in accordance with the method described in Toyamaand Ando, “Monoclonal Antibody Experiment Manual”, published byKodansha, 1987; Andou and Chiba, “Monoclonal Antibody ExperimentProcedure Manual”, published by Kodansha Scientific, 1991; Ishikawa,“Super High Sensitivity Enzyme Immuno Assay”, published byGakkai-syuppan center, 1993; Ed Harlow and David Lane, “Antibodies ALaboratory Manual”, published by Cold Spring Harbor Laboratory, 1988 andA. Doyle and J. B. Griffiths, “Cell & Tissue Culture: LaboratoryProcedures”, published by John Wiley & Sons Ltd., 1996. In some assays,the condition of reaction were modified, for example, the reaction wasperformed at 4° C. over night. Antibodies to human-immunogloblin orantigen were diluted to about 0.5-10 μg/ml (100-5000 fold) and ELISAplates were coated with these solutions. PBS supplemented with 5% mouseserum (Sigma, M5905) was used for blocking and dilution of the samplesand labeled antibodies. PBS was used for 20-fold dilution of thechimeric mouse sera. After washed, the coated plate was blocked over 1hour. After plate was washed, sample was added and incubated over a halfhour. After washed, Enzyme labeled anti-human immunogloblin antibodiesdiluted 100-5000 folds were added to the plates and incubated over 1hour, the plate was washed and then substrate was added. In some assays,the same procedure was applied except that a biotin-labeled antibody wasused. After plate was washed, avidin-enzyme complex was added. Afterplate was washed, substrate was added. Absorbances were measured with amicroplate reader (Bio-tek instrument, EL312e). The chimeric mice(Example 10, K3-1-2, K3-2-2 and K3-2-3) which were 29-35 days old werebled and assayed by ELISA. Anti-human IgM mouse monoclonal antibody(Sigma, I6385) was diluted with 50 mM carbonate-bicartonate buffer (pH9.6) and absorbed to the 96-well microtiter plates. The serum samplesdiluted with mouse serum (Sigma, M5905) supplemented PBS were added tothe plates. Subsequently, peroxidase-labeled anti-human IgM goatantibody (Tago, 2392) was added and the plates were incubated. AfterABTS substrate (Kirkegaard & Perry Laboratories Inc., 506200) was added,enzyme activity was determined by absorbance measurement at 405 nm.Purified human IgM antibody (CAPEL, 6001-1590) and IgG (Sigma, I4506)were used as standards. The standards were diluted stepwise with mouseserum-supplemented PBS. In the determination of human IgG concentration,anti-human IgG goat antibody (Sigma, I3382) was absorbed to the plateand the human IgG was detected with peroxidase-labeled anti-human IgGgoat antibody (Sigma, A0170). The results are shown in Table 5. Bothhuman IgM and IgG were detected. TABLE 5 Concentrations of HumanAntibodies in Chimeric Mouse Sera (ELISA) Chimeric Mouse IgG (mg/l) IgM(mg/l) K3-1-2 0.37 3.7 K3-2-2 0.33 5.9 K3-2-3 0.51 3.4

[0300] Two milliliters of human serum albumin (HSA, Sigma, A3782)dissolved in PBS was mixed with adjuvant (MPL+TDM Emulsion, RIBIImmunochem Research Inc.) to prepare an antigen solution at aconcentration of 0.25 mg/ml. The chimeric mice retaining humanchromosome #14 fragment (Example 10, K3-1-1 and K3-2-1) were immunizedwith 0.2 ml of the antigen solution 3 times at days 27, 34 and 41 afterbirth. The chimeric mouse sera were assayed by ELISA. The results areshown in FIGS. 13 and 14. The human antibody concentration in the seraof the HSA-immunized chimeric mice was increased after the immunization.In the K3-1-1 mouse, 18 μg/mi of human IgM and 2.6 μg/ml of IgG weredetected in the serum at day 17 after the immunization. In the serum ofthe control ICR mouse, the human antibody titer was not significant.

EXAMPLE 15

[0301] Production of Human Antibody Heavy Chain-Producing Hybridomasfrom the Human Chromosome #14 Transferred Chimeric Mouse

[0302] The spleen was removed from the human albumin-immunized chimericmouse (K3-1-1, Example 14) at day 44 after birth. The spleen cell wasfused with a myeloma cell to produce a hybridoma. The hybridoma wasproduced using a myeloma cell P3×63Ag8.653 (DAINIPPON PHARMACEUTICALCO., LTD., 05-565) by the method described in Ando and Chiba,“Monoclonal Antibody Experimental Procedure Manual”, published byKodansha Scientific, 1991. The hybridomas were inoculated into ten96-well plates and cultured for 1 week. The culture supernatant wasanalyzed by ELISA. The ELISA procedure was conducted by using anti-humanIgM mouse monoclonal antibody (Sigma, I6385) immobilized on ELISA platein the same manner as in Example 14 to give 6 positive clones. HSA(antigen) was dissolved in 50 mM carbonate-bicarbonate buffer (pH 9.6)at a concentration of 5 μg/ml and the antigen solution was dispensed in100 μl portions into all the wells of the ELISA plates. After theaddition of the supernqtant, peroxidase-labeled anti-human IgA+IgG+IgMgoat antibodies (Kierkegaard & Perry Laboratories Inc., 04-10-17) wereused for detection of HSA-specific human antibody. One positive clonewas confirmed in the ten plates. This clone was one of the 6 human IgMpositive clones. The clone (H4B7) was further cultured and the culturesupernatant was diluted, followed by ELISA analysis using HSA as anantigen with peroxidase-labeled anti-human IgM goat antibody (Tago,2392) in the same manner as described above. As a result, the absorbancedecreased with the increase in the dilution of the culture solution.Serial twofold dilutions of 2 μg/ml human IgM (CAPEL, 6001-1590) showedlow absorbance regardless of dilution ratios. This suggests that theantibody produced by hybridoma H4B7 had a specificity to HSA (FIG. 15).In FIG. 15, the dilution of the culture supernatant samples is plottedon the horizontal axis and the absorbance at 405 nm is plotted on thevertical axis.

EXAMPLE 16

[0303] Re-Marking of the G418 Resistance-Marked Human Chromosome #2Fragment with Puromycin Resistance

[0304] The A9 cells retaining the G418 resistance-marked humanchromosome #2 fragment (W23) (see Example 1, FIG. 1) were cultured in aG418 (800 μg/ml) containing selective medium (10% FBS, DMEM) in a 100 mmplate. Plasmid pPGKPuro (provided by Dr. Peter W. Laird (WHITEHEADINSTITUTE)) containing puromycin resistance gene was linearized withrestriction enzyme SalI (TAKARA SHUZO CO., LTD.) before transfection.The cells were treated with trypsin and suspended in Dulbecco'sphosphate buffered saline (PBS) at a concentration of 5 x 106 cells/ml,followed by electroporation using a Gene Pulser (Bio-Rad Laboratories,Inc.) in the presence of 10 μg of DNA in the same manner as inExample 1. A voltage of 1000 V was applied at a capacitance of 25 μFwith an Electroporation Cell of 4 mm in length (Example 1) at roomtemperature. The electroporated cells were inoculated into media in 3-6plates of 100 mmφ. After one day, the medium was replaced with adouble-selective medium containing 10 μg/ml of puromycin (Sigma, P-7255)and 800 μg/ml of G418. The colonies formed after 2-3 weeks werecollected in groups each consisting of about 200 colonies. The cells ofeach of the three groups were cultured in two or three 25 cm² flasks toform microcells. The mouse A9 cells were cultured in a 25 cm² flask andfused with the microcells by the same procedure as in Example 1. Thefused cells were transferred into two 100 mm plates and cultured in thedouble-selective medium containing G418 and puromycin. One of the threegroups gave two double-drug resistant clones. In these clones, it wasmost likely that puromycin resistance marker had been introduced intohuman chromosome #2 fragment.

EXAMPLE 17

[0305] Duplication of Transferred Human Chromosome in the HumanChromosome Transferred ES Cells

[0306] The ES cell clone retaining the G418 resistance marked humanchromosome #14 fragment (E14/#14-36) was cultured in a medium containingG418 at a high concentration to give ES cell clones in which the humanchromosome was duplicated (“Biomanual Series 8, Gene Targeting”,published by Yodosha, 1995). G418 resistant mouse primary cells(purchased from Lifetech Oriental) were inoculated into a 100 mm platewithout treating with mitomycin C and used as feeder cells. TheE14/#14-36 cells were inoculated into the 100 mm plate and after half aday, the medium was replaced with a medium containing G418 at aconcentration of 16 mg/ml. The medium was replaced every 1-2 days. TheG418 concentration was changed to 10 mg/ml one week later and thecultivation was continued. Among the colonies formed, 15 were picked upand cultured, followed by FISH analysis of chromosome using humanchromosome #14 specific probes (see Example 9). As a result, humanchromosome #14 fragment was found to have duplicated in the 8 clones.

EXAMPLE 18

[0307] Preparation of Mouse ES Cells Retaining Both Human Chromosome #2Partial Fragments and Human Chromosome #14 Partial Fragments.

[0308] In a microcell transfer experiment using the double-drugresistant clone PG-1 from Example 16 as a microcell donor cell and awild-type A9 cell as a recipient cell, it was confirmed that the humanchromosome #2 partial fragment retained in PG-1 was marked with apuromycin resistance gene. The preparation of microcells and the fusionwith the A9 cells was carried out by the same methods as in Example 1.As a result, 10 days after the microcell fusion, a total of fifty nineG418 resistant colonies appeared. After the medium for these colonieswas changed to one containing 8 μg/ml puromycin, the colonies werecultured for 3 days to give 45 viable colonies (76%). In many cases ofmicrocell fusion, only one or few chromosomes are transferred into arecipient cell. Hence, cotransfer of both the resistance genes at a highfrequency shows that the G418 resistance-labeled chromosome #2 partialfragment retained in the PG1 clone was also marked with the puromycinresistance gene. In addition, for the detection of the respective markergenes on the human chromosome #2 partial fragment, FISH analysis wasconducted by using pSTneoB (see Example 1) as a probe in the case of theA9/∩2 W23 clone having only G418 resistance (see Example 16) and byusing pPGKPuro (see Example 16) as a probe in the case of the PG1 clonein accordance with the method described in Matsubara et al., “FISHExperiment Protocol”, published by Shujunsha, 1994. As a result, in thecase of the A9/∩2 W23 clone, one signal was observed in each of thesister chromatids of the human chromosome #2 partial fragment observedin Example 12 (2 signals in total). This indicated the insertion ofpSTneoB into the human chromosome #2 partial fragment at one site. Inthe case of the PG1 clone, a total of 4 signals were observed on achromosome fragment of the same size as in A9/∩2 W23. Since pSTneoB andpPGKPuro had identical sequences in their vector portions, the pSTneoBcould be detected by the pPGKPuro probe. Hence, it is believed that outof the four signals observed in the PG1 clone, two were from the pSTneoBand the other two were from the pPGKPuro. These results show that thehuman chromosome #2 partial fragment retained in the PG1 was marked withboth the G418 and puromycin resistances.

[0309] The PG1 cell clone was used as a chromosome donor cell to preparea mouse ES cell retaining both a human chromosome #2 partial fragmentand a human chromosome #14 partial fragment. The G418 resistant TT2 cellclone 1-4 already retaining the human chromosome #14 partial fragment(see Example 9) was used as a chromosome recipient cell. The microcellfusion and the selection of puromycin resistant cells were carried outby the same methods as in the selection of the G418 resistant clones inExample 9 except that the concentration of puromycin was 0.75 μg/ml. Thefrequency of the appearance of the resulting puromycin resistant cloneswas 3-7 per 10⁷ of 1-4 cells. The presence of G418 resistance in thesepuromycin resistant clones was confirmed from the fact that they weregrown in the presence of 300 μg/ml of G418. The double-drug resistantclones were stored frozen and genomic DNA was prepared by the samemethods as in Example 2. The retention of the human chromosome #2partial fragment and human chromosome #14 partial fragment was confirmedby the method described in (1) in the case of double-drug resistantclones PG5, PG15 and PG16 and by the method described in (2) in the caseof the clone PG15.

[0310] (1) PCR Analysis

[0311] Genomic DNAs of the double-drug resistant clones were used astemplates in the PCR amplifications. Among the markers on humanchromosomes #2 and #14 (Genetic Maps, supra), the primers whose presencein the A9/∩2 W23 clone was confirmed in Example 12 and those whosepresence in the TT2/#14 1-4 clone was confirmed in Example 9 were used.All the primers gave expected amplification products in all the threeclones.

[0312] (2) Fluorescence in situ Hybridization (FISH)

[0313] FISH analysis was conducted by using FITC-labeled human total DNAas a probe in the same manner as in Example 11. As a result, in almostall of the metaphase spreads, two (large and small) human chromosomefragments were detected. The large fragment had the same size as that ofthe partial fragment detected by using the human chromosome #14 specificprobes in the case of the TT2/#14 1-4 clone in Example 9 and the smallfragment had the same size as that of the partial fragment detected byusing the human chromosome #2 specific probes in the case of the TT2/∩25-1 in Example 12. The results are shown in FIG. 16. In FIG. 16, theless bright chromosome was derived from the mouse. The two (large andsmall) chromosome fragments of high brightness due to FITC fluorescenceas shown by arrows were derived from the human, which are believed tocorrespond to the human chromosome #14 and #2 partial fragments.

[0314] These results show that the obtained double-drug resistant ESclones retained both the human chromosome #2 partial fragment and thehuman chromosome #14 partial fragment.

EXAMPLE 19

[0315] Production of Chimeric Mice from the Mouse ES Cell ClonesRetaining Both Human Chromosome #2 Partial Fragments and HumanChromosome #14 Partial Fragments

[0316] The cells in frozen stocks of the G418 and puromycindouble-resistant TT2 cell clones PG5, PG15 and PG16 from Example 18which were confirmed to retain human chromosome #2 partial fragments andhuman chromosome #14 partial fragments were thawed, started to cultureand injected into 8-cell stage embryos obtained by mating ICR orMCH(ICR) male and female mice (CREA JAPAN, INC.); the injection rate was10-12 cells per embryo. The embryos were cultured in a medium for EScells (see Example 9) overnight to develop to blastocysts. Two and ahalf day after a foster mother ICR mouse was subjected to apseudopregnant treatment, about ten of the injected embryos weretransplanted to each side of the uterus of the foster mother. Theresults are shown in Table 6. TABLE 6 Production of chimeric mice fromthe mouse ES cell clones retaining both human chromosome #2 partialfragments and human chromosome #14 partial fragments Number of ES EScell Double-drug cell-injected Number of Number of Contributionclone/human resistant 8-cell stage offspring chimeric to coat colorchromosome clone No. embryos mice mice <10% 10-50% 50%< TT2/#14 + #2 PG5160 26 8 7 1 — PG15 168 15 3 1 2 — PG16 223 32 12 3 6 3

[0317] As a result of the transplantation of a total of 551 injectedembryos, 73 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 73produced offsprings, 23 mice were recognized to have a partial agouticoat color, indicating the contribution of the ES cells.

[0318] These results show that the ES cell clones PG5, PG15 and PG16retaining human chromosome #2 partial fragments and human chromosome #14partial fragments maintain the ability to produce chimera, that is, theability to differentiate into normal tissues of mouse.

EXAMPLE 20

[0319] Detection of Human Antibody in sera of the Chimeric Mice Derivedfrom the ES Cells Retaining Both Human Chromosome #2 Partial Fragmentsand Human Chromosome #14 Partial Fragments

[0320] The two KPG-15 (9 weeks old; derived from the PG-5 clone, 10%chimerism) and KPG-18 (5 weeks old; derived from the PG-5 clone, 10%chimerism) chimeric mice from Example 19 were immunized with 0.2 ml of asolution of human serum albumin (HSA, Sigma, A3782) and adjuvant(MPL+TDM Emulsion, RIBI Immunochem Research Inc.) at a HSA concentrationof 0.25 mg/ml. The chimeric mice were bled just before the immunizationand 8 days after that and the concentrations of human antibody μ and κchains in the sera were determined by ELISA (see Example 14). Ninetysix-well microtiter plates were coated with anti-human antibody κ chaingoat antibody (VECTOR LABORATORIES INC., AI-3060) diluted with 50 mMcarbonate-bicarbonate buffer (pH 9.6) and then a serum sample dilutedwith mouse serum (Sigma, M5905)-containing PBS was added. Subsequently,biotin-labeled anti-human antibody κ chain goat antibody (VECTORLABORATORIES INC., BA-3060) was added to the plates and incubated. Acomplex of biotinylated horseradish peroxidase and avidin DH (VECTORLABORATORIES, INC., Vectastain ABC Kit, PK4000) was added and incubated.After 3,3′,5,5′-tetramethylbenzidine (TMBZ, Sumitomo Bakelite, ML-1120T)was added as a peroxidase substrate, enzyme activity was determined byabsorbance measurement at 450 nm. Purified human IgG antibody having Kchain (Sigma, I-3889) was used as standard. The standard was dilutedstepwise with mouse serum-supplemented PBS. In the case of μ chain,96-well microtiter plates were coated with anti-human antibody μ chainmouse monoclonal antibody (Sigma, I-6385) diluted with 50 mMcarbonate-bicarbonate buffer (pH 9.6) and then a serum sample was added.Subsequently, peroxidase-labeled anti-human antibody μ chain mouseantibody (The Binding Site Limited, MP008) was added to the plates andincubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added, enzymeactivity was determined by absorbance measurement at 450 nm. Purifiedhuman IgM antibody having μ chain (CAPPEL, 6001-1590) was used asstandard. The standard was diluted stepwise with mouseserum-supplemented PBS. As a result, both the human antibody μ and κchains were detected in both individuals. the concentrations of thesehuman antibodies in the sera increased after the immunization (Tables 7and 8). TABLE 7 Concentrations of Human Antibodies in Chimeric MouseKPG15 (ELISA) IgM (mg/l) Ig κ (mg/l) Before Immunization 0.19 1.6 8 DaysAfter Immunization 0.75 1.7

[0321] TABLE 8 Concentrations of Human Antibodies in Chimeric MouseKPG18 (ELISA) IgM (mg/l) Ig κ (mg/l) Before Immunization 0.29 0.57 8Days After Immunization 3.4 0.87

[0322] These results show that human antibody heavy and light chaingenes can function in the chimeric mice derived from the ES cellsretaining both human chromosome #2 partial fragments and humanchromosome #14 partial fragments.

EXAMPLE 21

[0323] Detection of Anti-HSA Human Antibody 7 Chain in sera of the HumanChromosome #14 Fragments Transferred Chimeric Mice

[0324] The chimeric mice retaining human chromosome #14 fragments whichwere produced by the same method as in Example 10 (K9 and K11: both werederived from the TT2 cell clone 3-2, with chimerisms of 50% and 30%,respectively) were immunized with HSA either 4 times at days 79, 93, 107and 133 after birth (K9) or 3 times at days 74, 88 and 111 after birth(K11) by the same method as in Example 20. Antibodies including human γchain against human serum albumin in the sera of the chimeric mice weredetected by ELISA. Ninety six-well microtiter plates were coated withHSA (Sigma, A 3782) diluted with 50 mM carbonate-bicarbonate buffer (pH9.6) and then a sample diluted with PBS was added. Subsequently,peroxidase-labeled anti-human IgG mouse antibody (Pharmingen, 08007E)was added to the plates and incubated. After O-phenylenediamine (OPD,Sumitomo Bakelite, ML-11300) was added as a peroxidase substrate, enzymeactivity was determined by absorbance measurement at 490 nm. The titerof the anti-HSA human IgG in the sera of the chimeric mice immunizedwith HSA increased after the immunization. On the other hand, controlICR mouse gave a background level of the anti-HSA human IgG titer afterthe immunization with HSA. The results are shown in FIG. 17. In FIG. 17,the number of days after the first immunization of the chimeric micewith HSA is plotted on the horizontal axis and the absorbance at 490 nmis plotted on the vertical axis. These results show that the antibodytiter of the antigen specific human IgG was increased by stimulationwith the HSA antigen in the chimeric mice retaining human chromosome #14fragments.

EXAMPLE 22

[0325] Detection of Human Antibody κ Chain in a Serum of the HumanChromosome #22 Fragment Transferred Chimeric Mouse

[0326] The chimeric mouse K22-7 from Example 3 (9 weeks old; 10%chimerism) was bled and human antibody λ chain in the serum was detectedby ELISA (see Example 14). Ninety six-well microtiter plates were coatedwith anti-human antibody λ chain goat antibody (VECTOR LABORATORIESINC., AI-3070) diluted with 50 mM carbonate-bicarbonate buffer (pH 9.6)and then a serum sample was added. Subsequently, biotin-labeledanti-human antibodyλ chain goat antibody (VECTOR LABORATORIES INC.,BA-3070) was added to the plates and incubated. A complex ofbiotinylated horseradish peroxidase and avidin DH (VECTOR LABORATORIES,INC., Vectastain ABC Kit) was added and incubated. After TMBZ (SumitomoBakelite, ML-1120T) was added as a peroxidase substrate, enzyme activitywas determined by absorbance measurement at 450 nm. Purified human IgGantibody having λ chain (Sigma, I-4014) was used as standard. Thestandard was diluted stepwise with mouse serum-supplemented PBS. As aresult, human antibody A chain was detected in the chimeric mouse at aconcentration corresponding to 180 ng/ml of human IgG. These resultsshow that human antibody λ chain gene can function in the chimeric mouseretaining a human chromosome #22 fragment.

EXAMPLE 23

[0327] Detection of Human Antibody κ Chain in sera of the HumanChromosome #2 Fragment Transferred Chimeric Mice

[0328] The chimeric mouse K2-8 from Example 13 (5 weeks old; 70%chimerism) and the chimeric mice K2-3, K2-4 and K2-12 from Example 13 (9weeks old; chimerisms was 50%, 20% and 80%, respectively) were bled andhuman antibody κ chain in the sera was detected by ELISA (see Example14). Ninety six-well microtiter plates were coated with anti-humanantibody κ chain goat antibody (VECTOR LABORATORIES INC., AI-3060)diluted with 50 mM carbonate-bicarbonate buffer (pH 9.6) and then aserum sample was added. Subsequently, biotin-labeled anti-human antibodyκ chain goat antibody (VECTOR LABORATORIES INC., BA-3060) was added tothe plates and incubated. A complex of biotinylated horseradishperoxidase and avidin DH (VECTOR LABORATORIES, INC., Vectastain ABC Kit)was added and incubated. After TMBZ (Sumitomo Bakelite, ML-1120T) wasadded, enzyme activity was determined by absorbance measurement at 450nm. Purified human IgG antibody having κ chain (Sigma, I-3889) was usedas standard. The standard was diluted stepwise with mouseserum-supplemented PBS. The results are shown in Table 9. TABLE 9Concentration of Human Antibody κ Chain in Chimeric Mouse (ELISA)Chimeric Mouse Ig κ (mg/l) K2-3 124 K2-4 85 K2-8 25 K2-12 56

[0329] The chimeric mice K2-3 and K2-4 retaining human chromosome #2fragments from Example 13 were immunized with HSA, 3 times at days 66,80 and 102 after birth by the same method as in Example 20. The chimericmouse K2-12 was immunized with HSA, 4 times at days 63, 77, 91 and 116after birth by the same method as in Example 20. Human antibody K chainagainst HSA in the sera of the chimeric mice was detected by ELISA (seeExample 14). Ninety six-well microtiter plates were coated with HSA(Sigma, A 3782) diluted with 50 mM carbonate-bicarbonate buffer (pH 9.6)and then a sample was added. Subsequently, biotin-labeled anti-humanantibody K chain goat antibody (VECTOR LABORATORIES, INC., BA-3060) wasadded to the plates and incubated. A complex of biotinylated horseradishperoxidase and avidin DH (VECTOR LABORATORIES, INC., Vectastain ABC Kit)was added and incubated. After OPD (Sumitomo Bakelite, ML-11300) wasadded as a peroxidase substrate, enzyme activity was determined byabsorbance measurement at 490 nm. The titer of the anti-HSA human κchain in the sera of the chimeric mice immunized with HSA increasedafter the immunization. On the other hand, control ICR mouse gave abackground level of the anti-HSA human κ chain titer after theimmunization. The results are shown in FIG. 18. In FIG. 18, the numberof days after the first immunization of the chimeric mice with HSA isplotted on the horizontal axis and the absorbance at 490 nm is plottedon the vertical axis. These results show that human antibody κ chaingene can function in the chimeric mice retaining human chromosome #2fragments and that the antibody titer of the antigen specific human Ig κwas increased by stimulation with the HSA antigen in the chimeric mice.

EXAMPLE 24

[0330] Preparation of Human Antibody Heavy Chain (μ chain or γchain)—Producing Hybridomas from the Human Chromosome #14 TransferredChimeric Mouse

[0331] The spleen was removed from the HSA-immunized chimeric mouse K9(see Example 21) at day 136 after birth. A spleen cell was fused with amyeloma cell to produce a hybridoma. The hybridoma was produced using amyeloma cell Sp-2/0-Ag14 (Dainippon Pharmaceutical Co., Ltd., 05-554) bythe method described in Toyama and Ando, “Monoclonal Antibody ExperimentProcedure Manual”, published by Kodansha Scientific, 1991. The cellswere inoculated into a medium containing 10% ORIGEN Hybridoma CloningFactor (HCF, Bokusui Brown) in eight 96-well plates and G418 was addedafter 3 days at a concentration of 1 mg/ml, followed by cultivation for1-3 weeks. The culture supernatant was analyzed by ELISA. Ninetysix-well microtiter plates were coated with anti-human μ chain mousemonoclonal antibody (Sigma, I-6385) diluted with 50 mMcarbonate-bicarbonate buffer (pH 9.6) and a sample diluted with PBS wasadded. Subsequently, peroxidase-labeled anti-human μ chain mouseantibody (The Binding Site LIMITED, MP008) was added to the plates andincubated. 2,2′-Azino-di-(3-ethyl-benzothiazoline-6-sulfonate)diammonium salt (ABTS, Kirkegaard & Perry Laboratories Inc., 04-10-17)was used as a substrate to detect seven positive clones. In thedetection of γ chain-producing clones, 96-well microtiter plates werecoated with anti-human γ chain mouse monoclonal antibody (Sigma, I-6260)and a sample diluted with PBS was added. Subsequently,peroxidase-labeled anti-human γ chain mouse antibody (Pharmingen,08007E) was added to the plates and incubated. ABTS (Kirkegaard & PerryLaboratories Inc., 04-10-17) was used as a substrate and two humanantibody γ chain-positive clones were obtained.

EXAMPLE 25

[0332] Preparation of Human Antibody Light Chain-Producing Hybridomasfrom the Human Chromosome #2 Transferred Chimeric Mouse

[0333] The spleen was removed from the HSA-immunized chimeric mouse K2-3(see Example 23) at day 105 after birth. A spleen cell was fused with amyeloma cell to produce a hybridoma. The hybridoma was produced using amyeloma cell P3×63Ag8.653 (Dainippon Pharmaceutical Co., Ltd., 05-565)by the method described in Toyama and Ando, “Monoclonal AntibodyExperiment Procedure Manual”, published by Kodansha Scientific, 1991.The cells were inoculated into a medium containing 10% HCF (BokusuiBrown) in ten 96-well plates and G418 was added after 3 days at aconcentration of 1 mg/ml, followed by cultivation for 1-3 weeks. Theculture supernatant was assayed by ELISA. The ELISA analysis wasconducted by the same method as in Example 23 and two human antibody κchain-positive clones were obtained.

EXAMPLE 26

[0334] Re-Marking of the G418 Resistance-Marked Human Chromosome #22with Puromycin Resistance

[0335] The A9 cells retaining the G418 resistance-marked humanchromosome #22 (A9/#22 γ 2) from Example 1 were re-marked with puromycinresistance by the same method as in Example 16. About 200 colonies ofdouble-drug resistant clones obtained by electroporation of the γ 2cells with pPGKPuro were collected as one group and three such groups(P1, P2 and P3) were used as donor cells to perform microcell transferinto wild-type mouse A9 cells. As a result, 6, 1 and 3 of double-drugresistant clones were obtained from the groups P1, P2 and P3,respectively. The clone 6-1 from group P3 was used as a microcell donorcell and a wild-type A9 cell as a recipient cell to perform a microcelltransfer experiment (see Example 18). As a result, the human chromosome#22 was confirmed to have been further marked with a puromycinresistance gene. The preparation of microcells and the fusion with A9cells were conducted by the same methods as in Example 1. As a result,twenty eight G418 resistant colonies appeared 11 days after themicrocell transfer. After the medium for these colonies was changed toone containing 8 μg/ml puromycin, these colonies were cultured for 3days to give 21 (75%) viable colonies. In many cases of microcellfusion, only one or few chromosomes are transferred into a recipientcell. Hence, cotransfer of both the resistance genes at a high frequencyshows that the G418 resistance-labeled chromosome #22 retained in the6-1 clone was marked with the puromycin resistance gene.

EXAMPLE 27

[0336] Preparation and Sequencing of cDNA of a Human Antibody HeavyChain Variable Region from the Human Antibody Heavy Chain-ProducingHybridoma

[0337] Among the human antibody heavy chain (IgM)-producing hybridomasobtained in Example 15, H4B7 (HSA-specific) and H8F9 (non-specific)hybridomas were selected. Total RNAs were obtained from these hybridomasusing ISOGEN (Nippon Gene). The synthesis of cDNA from 5 μg each of thetotal RNAs was conducted with a Ready-To-Go T-primed 1st strand Kit(Pharmacia Co.). Using the resulting cDNA and the following primersprepared with reference to Larrick et al., BIO/TECHNOLOGY, 7, 934-,1989; Word et al., Int. Immunol., 1, 296-, 1989, PCR was performed toamplify a human antibody heavy chain variable region. CM1 (human IgMconstant region): 5′-TTGTATTTCCAGGAGAAAGTG (SEQ ID NO:45) CM2 (ditto):5′-GGAGACGAGGGGGAAAAGGG (SEQ ID NO:46) HS1 (human heavy chain variableregion): 5′-ATGGACTGGACCTGGAGG(AG) (SEQ ID NO:47) TC(CT)TCT(GT)C (amixture of 8 sequences) HS2 (ditto):5′-ATGGAG(CT)TTGGGCTGA(GC)CTGG(GC)TTT(CT)T (SEQ ID NO:48) (a mixture of16 sequences) HS3 (ditto):5′-ATG(AG)A(AC)(AC)(AT)ACT(GT)TG(GT)(AT)(GCT)C(AT)(CT) (SEQ ID NO:49)(GC)CT(CT)CTG (a mixture of 6144 sequences) * ( ) means that any one ofthe bases therein should be selected.

[0338] In both cases of the H4B7 and H8F9 hybridomas, the first run ofPCR was performed by using three kinds of primer combinations ofHS1×CM1, HS2×CM1 and HS3×CM1 in 40 cycles at 94° C. for 1 minute, 50° C.for 2 minutes and 72° C. for 3 minutes with a Thermal Cycler 140(Perkin-Elmer Corp.). The PCR products were amplified again under thesame temperature conditions in 30 cycles using HS1l×CM2, HS2×CM2 andHS3×CM2 primers, respectively. The amplification products wereelectrophoresed on a 1.5% agarose gel and detected by staining withethidium bromide. As a result, an amplification product of about 490 bpwas detected with the HS3×CM2 primer in the case of the H4B7 hybridoma.In the case of the H8F9 hybridoma, a slight band was detected at thesame site with the HS3×CM2 primer. The band in the case of H8F9 wasamplified again with the HS3×CM2 primer in 30 cycles under the sametemperature conditions as above. As a result, the amplification productwas detected as a very intensive signal. These PCR products were clonedinto a pBlueScriptII SK+(Stratagene Ltd.) at a SmaI site in accordancewith the method described in Ishida et al., “Gene Expression ExperimentManual”, published by Kodansha Scientific, 1995. Among the amplificationproduct-inserted plasmids, plasmids #2, #3, #4(H4B7), #11, #13 and #14(H8F9) were selected and the nucleotide sequences of the amplificationproducts were determined with a Fluorescence Autosequencer (AppliedBiosystems Inc.). As a result of the comparison of the obtainednucleotide sequences or deduced amino acid sequences with those of knownhuman antibody VH region (Marks et al., Eur. J. Immunol. 21, 985-, 1991)and JH region (Ravetch et al., Cell, 27, 583-, 1981), it was revealedthat both the H4B7 and H8F9 hybridomas contained a combination of genesfor VH4 family and JH2. These results show that the chimeric mouseretaining human chromosome #14 partial fragment produced a completefunctional human antibody heavy chain protein.

EXAMPLE 28

[0339] Preparation and Sequencing of cDNA of Human Antibody κ Chain fromthe Spleen of the Human Antibody κ Chain-Expressing Chimeric Mouse

[0340] In the same manner as in Example 5, cDNA was prepared from thespleen of the chimeric mouse K2-8 from Example 13 which was confirmed toexpress human antibody κ chain in Example 23. Using the resulting cDNAand the following primers prepared with reference to Larrick et al.BIO/TECHNOLOGY, 7, 934-, 1989; Whitehurst et al., Nucleic Acids Res.,20, 4929-, 1992, PCR was performed to amplify human antibody κ chainvariable region. cDNA from the liver of the chimeric mouse K2-8 and cDNAfrom the spleen of the chimeric mouse K3-2-2 derived from the TT2/#143-2 clone (see Example 10) were used as negative controls. KC2 (human Igκ chain constant region): 5′-CAGAGGCAGTTCCAGATTTC (SEQ ID NO:50) KC3(ditto): 5′-TGGGATAGAAGTTATTCAGC (SEQ ID NO:51) KVMIX (human Ig κ chainvariable region):5′-ATGGACATG(AG)(AG)(AG)(AGT)(CT)CC(ACT)(ACG)G(CT)(GT)CA(CG)CTT (SEQ IDNO:52) (a mixture of 3456 sequences) * ( ) means that any one of thebases therein should be selected.

[0341] PCR was performed by using primer combinations of KVMIX×KC2 andKVMIX×KC3 in 40 cycles at 94° C. for 15 seconds, 55° C. for 15 secondsand 72° C. for 20 seconds with a Thermal Cycler 9600 (Perkin-ElmerCorp.). The amplification products were electrophoresed on a 1.5%agarose gel and detected by staining with ethidium bromide. As a result,expected amplification products of about 420 bp (KC2) and about 450 bp(KC3) were detected. In the case of the two negative controls, nospecific amplification product was detected. These amplificationproducts were cloned into a pBlueScriptII SK+ (Stratagene Ltd.) at aSmaI or EcoRI site in accordance with the method described in Ishida etal., “Gene Expression Experiment Manual”, published by KodanshaScientific, 1995. Among the amplification product-inserted plasmids,VK-#1 clone derived from the KVMIX×KC2 primers was selected and thenucleotide sequence of the amplification product was determined with aFluorescence Autosequencer (Applied Biosystems Inc.). Since the obtainednucleotide sequence did not contain a termination codon at any sitebetween an initiation codon and a constant region of human Igκ chain,the cloned amplification products are believed to encode a variableregion of functional human Igκ chain. As a result of the comparison ofthe obtained nucleotide sequences with those of known human antibody Vκregion (Klein et al., Eur. J. Immunol. 23, 3248-, 1993) and Jκ region(Whitehurst et al., supra), it was revealed that the VK-#1 clonecontained a combination of genes for Vκ 3 family and Jκ 4. These resultsshow that the chimeric mouse retaining human chromosome #2 partialfragment produced a complete functional human antibody κ chain protein.

EXAMPLE 29

[0342] Detection and Quantitation of Human Antibody γ Chain Subclassesand μ Chain in sera of the Chimeric Mice Retaining Human Chromosome #14Fragment

[0343] The chimeric mice K15A and K16A from Example 10 (derived from the1-4 clone, with chimerism of 70% and 50%, respectively) of 11 weeksafter birth were bled and human antibody γ chain subclasses and μ chainin the sera were detected by the same ELISA method as in Example 14.

[0344] Quantative Determination of Human IgG1

[0345] Ninety six-well microtiter plates were coated with anti-human IgGantibody (Sigma, I-6260) diluted with PBS. A serum sample was added.Subsequently, peroxidase-labeled anti-human IgG1 antibody (Pharmingen,08027E) was added to the plates and incubated. After TMBZ (SumitomoBakelite, ML-1120T) was added, enzyme activity was determined byabsorbance measurement at 450 nm. Purified human IgG1 antibody (Sigma,I-3889) was used as standard. The standard was diluted stepwise withmouse serum-supplemented PBS.

[0346] Quantative Determination of Human IgG2

[0347] Ninety six-well microtiter plates were coated with anti-humanIgG2 antibody (Sigma, I-9513) diluted with PBS. A serum sample wasadded. Subsequently, peroxidase-labeled anti-human IgG antibody (Sigma,A-0170) was added to the plates and incubated. After TMBZ (SumitomoBakelite, ML-1120T) was added, enzyme activity was determined byabsorbance measurement at 450 nm. Purified human IgG2 antibody (Sigma,I-4139) was used as standard. The standard was diluted stepwise withmouse serum-supplemented PBS.

[0348] Quantative Determination of Human IgG3

[0349] Anti-human IgG3 antibody (Sigma, I-7260) was diluted with 100 mMglycine-HCl buffer (pH 2.5) and incubated for 5 minutes at roomtemperature, followed by 10-fold dilution with 100 mM phosphate buffer(pH 7.0). Ninety six-well microtiter plates were coated with theanti-human IgG3 antibody solution. A serum sample was added.Subsequently, peroxidase-labeled anti-human IgG antibody (Pharmingen,08007E) was added to the plates and incubated. After TMBZ (SumitomoBakelite, ML-1120T) was added, enzyme activity was determined byabsorbance measurement at 450 nm. Purified human IgG3 antibody (Sigma,I-4389) was used as standard. The standard was diluted stepwise withmouse serum-supplemented PBS.

[0350] Quantative Determination of Human IgG4

[0351] Anti-human IgG4 antibody (Sigma, I-7635) was diluted with 100 mMglycine-HCl buffer (pH 2.5) and incubated for 5 minutes at roomtemperature, followed by 10-fold dilution with 100 mM phosphate buffer(pH 7.0). Ninety six-well microtiter plates were coated with theanti-human IgG3 antibody solution. A serum sample was added.Subsequently, peroxidase-labeled anti-human IgG antibody (Pharmingen,08007E) was added to the plates and incubated. After TMBZ (SumitomoBakelite, ML-1120T) was added, enzyme activity was determined byabsorbance measurement at 450 nm. Purified human IgG4 antibody (Sigma,I-4639) was used as standard. The standard was diluted stepwise withmouse serum-supplemented PBS.

[0352] Quantative Determination of Human IgM

[0353] Ninety six-well microtiter plates were coated with anti-human μchain mouse monoclonal antibody (Sigma, I-6385) diluted with PBS. Aserum sample was added. Subsequently, peroxidase-labeled anti-human μchain mouse antibody (The Binding Site Limited, MP008) diluted withmouse serum (Sigma, M5905)-supplemented PBS was added to the plates andincubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added as aperoxidase substrate, enzyme activity was determined by absorbancemeasurement at 450 nm. Purified human IgM having μ chain (CAPPEL,6001-1590) was used as standard. The standard was diluted stepwise withmouse serum (Sigma, M5905)-supplemented PBS. The results are shown inTable 10. All the subclasses IgG1, IgG2, IgG3 and IgG4, and IgM weredetected in the two chimeric mice K15A and K16A. TABLE 10 Concentrationsof Human antibody IgG Subclasses and IgM in the Chimeric Mice (ELISA)Chimeric mouse IgG1 IgG2 IgG3 IgG4 IgM (mg/l) K15A 2.25 1.96 0.17 0.437.09 K16A 0.30 0.69 0.10 0.07 0.87

EXAMPLE 30

[0354] Preparation of Mouse ES Cell Clones (TT2) Retaining HumanChromosome #22

[0355] The cell clone 6-1 (A9/#22, G418 and puromycin resistant) fromExample 26 was used as a chromosome donor cell for the preparation ofmouse ES cell (TT2) retaining human chromosome #22. A wild-type TT2 cellline (see Example 9) was used as a chromosome recipient cell. Themicrocell fusion and the selection of puromycin resistant clones wereconducted by the same procedures as in the selection of G418 resistantclones in Example 9 except that the concentration of puromycin was 0.75μg/ml. The frequency of the appearance of the puromycin resistant cloneswas 1-2 per 10⁷ of TT2 cells. The puromycin resistant clones were storedfrozen and genomic DNA was prepared by the same methods as in Example 2.The retention of human chromosome #22 in the puromycin resistant clonePG22-1 was confirmed by the methods described in (1) and (2) below.

[0356] (1) PCR Analysis

[0357] Genomic DNA of the puromycin resistant clone was used as atemplate in PCR amplification. Among the genes on human chromosome #22(Genetic Maps, supra), ten primers whose presence in the A9/#22 clonewas confirmed in Example 2 were used in the PCR amplification. All themarkers which existed in the A9/#22 clone (see Example 2) were detected.

[0358] (2) Southern Blot Analysis

[0359] In accordance with the same method as described in Example 2using human L1 sequence as a probe, Southern blot analysis was conductedwith genomic DNAs obtained from wild-type TT2 (negative control), thechromosome donor cell 6-1 and the puromycin resistant TT2 cell clonePG22-1. The results are shown in FIG. 19. In FIG. 19, the molecularweights of DNAs are shown at the left side. The band pattern of thePG22-1 clone was equivalent to that of the 6-1 cell and the signalintensities were the same. Hence, it was confirmed that chromosome #22in the 6-1 cell had been transferred certainly into the PG22-1 clone.

[0360] These experiments demonstrate that the puromycin resistant TT2cell clone PG22-1 retained the whole or the most part of humanchromosome #22.

EXAMPLE 31

[0361] Production of Chimeric Mice from the Mouse ES Cells (TT2)Retaining Human Chromosome #22

[0362] The cells in a frozen stock of the puromycin resistant TT2 cellclone PG22-1 from Example 30 which was confirmed to retain humanchromosome #22 were thawed, started to culture and injected into 8-cellstage embryos obtained by mating ICR or MCH(ICR) male and female mice(CREA JAPAN, INC.); the injection rate was 10-12 cells per embryo. Theembryos were cultured in a medium for ES cells (see Example 9) overnightto develop to blastocysts. Two and a half day after a foster mother ICRmouse was subjected to a pseudopregnant treatment, about ten of theinjected embryos were transplanted to each side of the uterus of thefoster mother. The results are shown in Table 11. TABLE 11 Production ofchimeric mice from the TT2 cell clone retaining human chromosome #22Number of ES ES cell Puromycin cell-injected Number of NumberContribution clone/human resistant 8-cell stage offspring chimeric tocoat color chromosome clone No. embryos mice mice <20% 20-50% 50-80%TT2/#22 PG22-1 266 36 8 4 1 3

[0363] As a result of the transplantation of a total of 266 injectedembryos, 36 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 36produced offsprings, 8 mice were recognized to have a partial agouticoat color, indicating the contribution of the ES cells.

[0364] These results show that the ES cell clone (derived from TT2,PG22-1) retaining human chromosome #22 maintain the ability to producechimera, that is, the ability to differentiate into normal tissues ofmouse.

EXAMPLE 32

[0365] Detection and Quantitation of Human Antibody λ Chain in sera ofthe Chimeric Mice Retaining Human Chromosome #22

[0366] The concentration of human antibody λ in the sera of the chimericmice KPG22-1, 2 and 3 from Example 31 was determined by ELISA inaccordance with the same procedure as in Example 14. The chimeric miceof 2 months after birth were bled and human antibody A chain in the serawas detected by ELISA. Ninety six-well microtiter plates were coatedwith anti-human immunoglobulin A chain antibody (VECTOR LABORATORIESINC., IA-3070) diluted with PBS and then a serum sample was added.Subsequently, biotin-labeled anti-human immunoglobulin λ chain antibody(VECTOR LABORATORIES INC., BA-3070) was added to the plates andincubated. A complex of biotinylated horseradish peroxidase and avidinDH (VECTOR LABORATORIES, INC., Vectastain ABC Kit) was added andincubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added, enzymeactivity was determined by absorbance measurement at 450 nm. Purifiedhuman IgM antibody having λ chain (Dainippon Pharmaceutical Co., Ltd.,U13200) was used as standard. The standard was diluted stepwise withmouse serum-supplemented PBS. The results are shown in Table 12. Theseresults show that human antibody λ chain gene can function in thechimeric mice retaining human chromosome #22. TABLE 12 Concentration ofHuman Antibody λ Chain in Chimeric Mice (ELISA) Chimeric Mouse %Chimerism Ig λ (mg/l) KPG22-1 50 12 KPG22-2 50 18 KPG22-3 20 24

EXAMPLE 33

[0367] Detection of Anti-Human HSA Human Antibody λ Chain in a Serum ofthe Human Chromosome #22 Transferred Chimeric Mouse

[0368] The chimeric mouse KPG22-3 from Example 31 was immunized withHSA, 3 times at days 79, 94 and 110 after birth by the same method as inExample 20. Human antibody λ chain in the serum of the chimeric mousewas detected by ELISA in accordance with the same procedure as inExample 14. Ninety six-well microtiter plates were coated with HSA(Sigma, A 3782) diluted with 50 mM carbonate-bicarbonate buffer (pH 9.6)to a concentration of 5 μg/ml and a serum sample was added. Biotinylatedanti-human Ig λ antibody (VECTOR LABORATORIES INC., BA-3070) was added.Subsequently, a complex of biotinylated-horseradish peroxidase andavidin DH (VECTOR LABORATORIES, INC., Vectastain ABC Kit) was added tothe plates and incubated. After TMBZ (Sumitomo Bakelite, ML-1120T) wasadded, enzyme activity was determined by absorbance measurement at 450nm. The titer of the anti-HSA human λ chain in the serum of the chimericmouse increased after the immunization. On the other hand, control ICRmouse gave a background level of the anti-HSA human λ chain titer afterthe immunization with HSA. The results are shown in FIG. 20. In FIG. 20,the number of days after the first immunization of the chimeric mousewith HSA is plotted on the horizontal axis and the absorbance at 450 nmis plotted on the vertical axis. These results show that human antibodyλ chain gene can function in the chimeric mouse retaining humanchromosome #22 and that the antibody titer of the antigen specific humanIg λ was increased by stimulation with the HSA antigen.

EXAMPLE 34

[0369] Preparation of Human Antibody Light Chain-Producing Hybridomasfrom the Human Chromosome #22 Transferred Chimeric Mouse

[0370] The spleen was removed from the mouse KPG22-3 (see Example 33) atday 113 after birth by the same method as in Example 25. A spleen cellwas fused with a myeloma cell to produce a hybridoma. The hybridoma wasproduced using a myeloma cell SP-2/0-Ag14 (Dainippon Pharmaceutical Co.,Ltd., 05-554) by the method described in Toyama and Ando, “MonoclonalAntibody Experiment Manual”, published by Kodansha Scientific, 1991. Thecells were inoculated into a medium containing 10% HCF (Air Brown) infive 96-well plates and cultured for 1-3 weeks. The supernatant of theculture solution in colony-positive wells was analyzed by ELISA. TheELISA analysis was conducted by the same method as in Example 33 andfour human antibody λ chain-positive clones were obtained.

EXAMPLE 35

[0371] Preparation of Mouse ES Cell Clones Retaining Both a HumanChromosome #22 Partial Fragment and a Human Chromosome #14 PartialFragment

[0372] The 6-1 cell clone from Example 26 (A9/#22, G418 and puromycinresistant) was used as a chromosome donor cell for the preparation ofmouse ES cells retaining both a human chromosome #22 partial fragmentand a human chromosome #14 partial fragment. The G418 resistant TT2 cellclone 1-4 retaining a human chromosome #14 partial fragment from Example9 was used as a chromosome recipient cell. The experiment of microcellfusion and the selection of puromycin resistant cells were carried outby the same methods as in the selection of the G418 resistant clones inExample 9 except that the concentration of puromycin was 0.75 μg/ml. Asa result, the frequency of the appearance of the puromycin resistantclones was 1-2 per 10⁷ of 1-4 cells. The retention of G418 resistance inthe puromycin resistant clones was confirmed from the fact that theseclones were grown in the presence of 300 μg/ml G418. The double-drugresistant clones were stored frozen and genomic DNAs were prepared bythe same methods as in Example 2. The retention of human chromosome #22and a human chromosome #14 partial fragment in the double-drug resistantclone PG22-5 was confirmed by PCR analysis. With genomic DNA of thedouble-drug resistant clone used as a template, PCR amplification wasconducted using primers whose presence on chromosome #22 was confirmedin Example 2 (A9/#22) and primers whose presence on chromosome #14 wasconfirmed in Example 9 (TT2/#14 1-4); as a result, three markers(D22S275, D22S315 and Ig λ) of the ten markers on chromosome #22 and allof the markers on chromosome #14 in the TT2/#14 1-4 clone were detected.

[0373] These experiments demonstrate that the obtained double-drugresistant TT2 cell clone retained both a human chromosome #22 partialfragment and a human chromosome #14 partial fragment.

EXAMPLE 36

[0374] Production of the Chimeric Mouse from the Mouse ES Cell CloneRetaining Both a Human Chromosome #22 Partial Fragment and a HumanChromosome #14 Partial Fragment

[0375] The cells in a frozen stock of the G418 and puromycindouble-resistant TT2 cell clone PG22-5 from Example 35 which wasconfirmed to retain a human chromosome #22 partial fragment and a humanchromosome #14 partial fragment were thawed, started to culture andinjected into 8-cell stage embryos obtained by mating ICR or MCH(ICR)male and female mice (CREA JAPAN, INC.); the injection rate was 10-12cells per embryo. The embryos were cultured in a medium for ES cells(see Example 9) overnight to develop to blastocysts. Two and a half dayafter a foster mother ICR mouse was subjected to a pseudopregnanttreatment, about ten of the injected embryos were transplanted to eachside of the uterus of the foster mother. The results are shown in Table13. TABLE 13 Production of the chimeric mouse from the mouse ES cellclone retaining both a human chromosome #22 partial fragment and a humanchromosome #14 partial fragment Number of ES cell- ES cell Double-druginjected Number of Number of Contribution clone/human resistant 8-cellstage offspring chimeric to coat color chromosome clone No. embryos micemice <20% 20-50% 50-80% TT2/#22 + #14 PG22-5 302 16 5 3 2 0

[0376] As a result of the transplantation of a total of 302 injectedembryos, 16 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 16produced offsprings, 5 mice were recognized to have a partial agouticoat color, indicating the contribution of the ES cell.

[0377] These results show that the ES cell clone PG22-5 retaining ahuman chromosome #22 partial fragment and a human chromosome #14 partialfragment maintains the ability to produce chimera, that is, the abilityto differentiate into normal tissues of mouse.

EXAMPLE 37

[0378] Detection of Human Antibody λ Chain and μ Chain in sera of theChimeric Mice Derived from the ES Cells Retaining Both a HumanChromosome #22 Partial Fragment and a Human Chromosome #14 PartialFragment

[0379] The chimeric mice KPG22-9, 10 and 12 from Example 36 wereimmunized with HSA. The chimeric mice KPG22-9 and 10 were immunized 11weeks after birth and bled 2 weeks after the immunization. The chimericmouse KPG22-12 was immunized twice at 7 and 11 weeks after birth andbled 2 weeks after the second immunization.

[0380] A serum human antibody μ chain, a serum human antibody λ chain,and a serum antibody having both human antibody λ and μ chains weredetected by ELISA in accordance with Example 14.

[0381] For the detection of complete human antibody molecules, 96-wellmicrotiter plates were coated with anti-human immunoglobulin λ chainantibody (Kirkegaard & Perry Laboratories Inc., 01-10-11) diluted withPBS and a serum sample was added. Subsequently, peroxidase-labeledanti-human immunoglobulin μ chain antibody (The Binding Site Limited,MP008) was added to the plates and incubated. After TMBZ (SumitomoBakelite, ML-1120T) was added as a peroxidase substrate, enzyme activitywas determined by absorbance measurement at 450 nm. Purified human IgMantibody having λ chain (Dainippon Pharmaceutical Co., Ltd., U13200) wasused as standard. The standard was diluted stepwise with mouseserum-supplemented PBS. Human antibody μ and λ chains were detected anddetermined quantitatively by ELISA in the same manner as in Examples 29and 32. The results are shown in Table 14. TABLE 14 Concentrations ofHuman Antibodies in Chimeric Mice (ELISA) Chimerism ES clone Chimericmouse (%) IgM (mg/l) Ig λ (mg/l) IgM, λ (mg/l) PG22-5 KPG22-9 30 2.549.9 0.043 PG22-5 KPG22-10 5 4.96 21.5 0.333 PG22-5 KPG22-12 40 3.71 7.00.048 3-2 K9 50 6.66 — <0.003 PG22-1 KPG22-2 50 — 17.6 <0.003

[0382] Both λ and μ chains were detected in the chimeric mice. Anantibody molecule having both human antibody μ and λ chains wasdetected. These results show: the human antibody λ chain gene and humanantibody μ chain gene can function at the same time in the chimeric micederived from the ES cells retaining human chromosome #22 partialfragments and human chromosome #14 partial fragments; and a completeantibody containing both human heavy and light chains was produced inpart of the B cells.

[0383] The control mice, that is, the chimeric mouse K9 retaining onlyhuman chromosome #14 from Example 10 and the chimeric mouse KG22-2retaining only human chromosome #22 from Example 31, gave backgroundlevels of an antibody having both human antibody λ and μ chains in thesera. It was confirmed that in these detection systems, only a completeantibody molecule having human λ and μ chains was detected.

EXAMPLE 38

[0384] Detection of Human Antibody having Human κ and μ Chains in seraof the Chimeric Mice Derived from the ES Cells Retaining Both HumanChromosome #2 Partial Fragments and Human Chromosome #14 PartialFragments

[0385] The chimeric mouse KPG-15 (derived from the TT2ES clone PG5, 10%chimerism) was immunized during 2-3 months after birth 3 times with 0.2ml of a solution of human serum albumin (HSA, Sigma, A3782) and adjuvant(MPL+TDM Emulsion, RIBI Immunochem Research Inc.) in PBS at a HSAconcentration of 0.25 mg/ml and bled (see Example 15). The chimericmouse KPG-26 (derived from the TT2ES clone PG6, 40% chimerism) of 6weeks after birth was bled. The concentration of a complete humanantibody molecule in the sera was determined by ELISA in accordance withExample 14. Ninety six-well microtiter plates were coated withanti-human immunoglobulin κ chain antibody (Kirkegaard & PerryLaboratories Inc., 01-10-10) diluted with PBS, and a serum sample wasadded. Subsequently, peroxidase-labeled anti-human immunoglobulin μchain antibody (The Binding Site Limited, MP008) was added to the platesand incubated. After TMBZ (Sumitomo Bakelite, ML-1120T) was added as aperoxidase substrate, enzyme activity was determined by absorbancemeasurement at 450 nm. Purified human IgM antibody having κ chain(CAPPEL, 6001-1590) was used as standard. The standard was dilutedstepwise with mouse serum-supplemented PBS. The concentrations of κchain and μ chain were determined by the same method as in Example 20.The results are shown in Table 15. TABLE 15 Concentrations of HumanAntibodies in Chimeric Mice (ELISA) Chimerism ES clone Chimeric mouse(%) IgM (mg/l) Ig κ (mg/l) IgM, κ (mg/l) PG-5 KPG15 10 0.18 1.01 0.075PG-6 KPG26 40 1.52 1.26 0.018 3-2 K9 50 6.66 — <0.002 5-1 K2-9 40 — 135<0.002

[0386] A antibody molecule having both human antibody μ and κ chains wasdetected. The control mice, that is, the chimeric mouse K9 retainingonly human chromosome #14 from Example 10 and the chimeric mouse K2-9retaining only human chromosome #2 from Example 13, gave backgroundlevels ( <0.002 mg/ml) of an antibody having human antibody κ and μchains in the sera. These results show: the human antibody κ chain geneand human antibody μ chain gene can function at the same time in thechimeric mice derived from the ES cells retaining both human chromosome#2 partial fragments and human chromosome #14 partial fragments; and acomplete antibody molecule containing both human heavy and light chainswas produced in part of the B cells.

EXAMPLE 39

[0387] Preparation of Mouse ES Cell Clone (TT2F, XO) Retaining a HumanChromosome #2 Partial Fragment

[0388] The cell clone PG1 from Example 16 was used as a chromosome donorcell for the preparation of a mouse ES cell (XO) retaining a humanchromosome #2 partial fragment. A TT2F cell (purchased from LifetecOriental Co.) having a karyotype of (39, XO), which was reported todifferentiate efficiently into an oocyte in chimeric mice (ShinichiAizawa, “Biomanual Series 8, Gene Targeting” published by Yodosha,1995), was used as a chromosome recipient cell. The experiment ofmicrocell fusion and the selection of puromycin resistant cells werecarried out by the same methods as in the selection of the G418resistant clones in Example 9 except that the concentration of puromycinwas 0.75 μg/ml. The frequency of the appearance of the puromycinresistant clones was 5 per 10⁷ of TT2F cells. The puromycin resistantclones were stored frozen and genomic DNAs were prepared from the clonesby the same methods as in Example 2. The retention of human chromosome#2 partial fragments in the drug resistant clones P-20 and P-21 wasconfirmed by PCR analysis. As a result of PCR amplification usinggenomic DNAs of the drug resistant clones as templates and three kindsof primers C κ, FABP1 and V κ 1-2 whose presence in the A9/∩2 W23 clonewas confirmed in Example 1, all of the three primers gave expectedamplification products in both of the two clones.

[0389] These experiments demonstrate that the obtained puromycinresistant ES cell clone (TT2F, XO) retained a human chromosome #2partial fragment.

EXAMPLE 40

[0390] Production of the Chimeric Mice from the Mouse ES Cell Clone(TT2F, XO) Retaining a Human Chromosome #2 Partial Fragment

[0391] The cells in a frozen stock of the puromycin resistant TT2F cellclone P-21 from Example 39 which was confirmed to retain a humanchromosome #2 partial fragment were thawed, started to culture andinjected into 8-cell stage embryos obtained by mating ICR or MCH(ICR)male and female mice (CREA JAPAN, INC.); the injection rate was 10-12cells per embryo. The embryos were cultured in a medium for ES cells(see Example 9) overnight to develop to blastocysts. Two and a half dayafter a foster mother ICR mouse was subjected to a pseudopregnanttreatment, about ten of the injected embryos were transplanted to eachside of the uterus of the foster mother. The results are shown in Table16. TABLE 16 Production of the chimeric mice from the TT2F cell cloneretaining a human chromosome #2 partial fragment Number of ES cell- EScell Puromycin injected Number of Number of Contribution clone/humanresistant 8-cell stage offspring chimeric to coat color chromosome cloneNo. embryos mice mice <20% 20-50% 50-90% 100% TT2F/#2fg. P-21 141 20 9 02 3 4

[0392] As a result of the transplantation of a total of 141 injectedembryos, 20 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2F cell-derived agouti coat color (darkbrown) in the host embryo-derived albino coat color. Out of the 20produced offsprings, 9 mice were recognized to have a partial agouticoat color, indicating the contribution of the ES cell. Four of the 9mice were chimeric mice having a full agouti coat color from the EScells.

[0393] These results show that the ES cell clone P-21 retaining a humanchromosome #2 partial fragment maintains the ability to produce chimera,that is, the ability to differentiate into normal tissues of mouse.

EXAMPLE 41

[0394] Detection and Quantitative Determination of Human Antibody κChain in sera of the Chimeric Mice Derived from the TT2F Clone Retaininga Human Chromosome #2 Partial Fragment

[0395] The chimeric mice K2-1F, 2F, 3F and 4F (derived from the P-21clone, 100% chimerism) from Example 40 of about 1 month after birth werebled and the concentration of human antibody κ chain in the sera wasdetermined quantitatively by ELISA in the same manner as in Example 20.

[0396] The results are shown in Table 17. It was confirmed that thehuman antibody κ chain gene could function in the chimeric mice when theTT2F was used as an ES cell. TABLE 17 Concentration of Human Antibody κChain in Chimeric Mice (ELISA) Chimeric mouse % Chimerism Ig κ (mg/l)K2-1F 100 66 K2-2F 100 156 K2-3F 100 99 K2-4F 100 20

EXAMPLE 42

[0397] Confirmation of the Retention of Human Chromosome in Progenies ofthe Chimeric Mice Derived from the Mouse ES Cell (TT2F, XO) Retaining aHuman Chromosome #2 Partial Fragment

[0398] Examination was made as to whether ES cell-derived progenieswould be reproduced by mating the female chimeric mice K2-1F and K2-4F(both were of 100% chimerism in coat color) from Example 40 with ICRmale mice. In such a mating, offspring mice of an agouti coat colorshould be reproduced from the oocytes derived from the TT2F cell (agouticoat color, dominant) in the chimeric mice and offspring mice of analbino coat color should be reproduced from oocytes derived from ICR ifthe oocytes are fertilized with the sperms from ICR male mice (albino,recessive). All the viable offspring mice (K2-1F, 10 mice and K2-4F, 5mice) obtained by one mating of the respective combinations had anagouti coat color which derived from the ES cells. The retention ofhuman chromosome fragments in genomic DNAs prepared from the tails ofthe offspring mice was examined by a PCR method. As a result of the PCRamplification using three kinds of primers whose presence in the P-21clone (see Example 39) was confirmed, the presence of these threemarkers was confirmed in 4 out of the ten mice from K2-1F and in 2 outof the five mice from K2-4F. The results of the PCR of these 15offspring mice are shown in FIG. 21. In FIG. 21, markers (Φ X174/HaeIIIfragment, Nippongene) and the DNA molecular weights of main bands areshown at the right side and the lengths of expected products ofamplification with the respective primers are shown by arrows at theleft side. At the right side, the results with tail-derived DNA of themother chimeric mice K2-1F and K2-4F (positive controls) are shown.These results show that the TT2 cell clone P-21 differentiated intofunctional oocyte in the chimeric mice and that a human chromosome #2partial fragment was transmitted to offsprings through oocyte.

EXAMPLE 43

[0399] Confirmation of the Retention of Human Chromosome in Progenies ofthe Chimeric Mice Derived from the Mouse ES Cell (TT2, XY) Retaining aHuman Chromosome #2 Partial Fragment

[0400] Examination was made as to whether ES cell-contributed offspringmice would be produced by mating K2-18 (70% chimeric male mouse fromExample 13) with K2-19 (60% chimeric female mouse of Example 13) ornon-chimeric female littermates. Since TT2 cell has the karyotype of(40, XY), it may differentiate into a functional sperm in the malechimeric mouse K2-18. If this is the case, offspring mice of an agouticoat color should be reproduced from ICR (albino, recessive)-derivedoocytes fertilized with sperms from TT2 cell (agouti color, dominant) inthe chimeric mice. While a total of 110 viable offspring mice wereobtained by the mating, ten had an agouti coat color which derived fromthe ES cells. The retention of human chromosome fragments in genomicDNAs prepared from the tails of 7 out of the ten offspring mice of anagouti coat color was examined by a PCR method. As a result of PCRamplification using two kinds of primers C κ and FABP1 whose presence inthe 5-1 clone (TT2/∩2fg. Example 12) was confirmed and primer V κ 1-2which was shown in Example 1, the presence of all of the three markerswas confirmed in 2 out of the seven mice. These results show that theTT2 cell clone 5-1 retaining a human chromosome #2 partial fragmentdifferentiated into functional sperms in the chimeric mice and that thehuman chromosome #2 partial fragment was transmitted to offspringsthrough the sperms.

EXAMPLE 44

[0401] Detection and Quantitative Determination of Human Antibody κChain in sera of Offspring Mice of the Chimeric Mice

[0402] The concentration of human antibody κ chain in the sera of theoffspring mice K2-1F-1˜10 and K2-4F-1˜5 from Example 42 was determinedquantitatively by ELISA. The mice of about 4-6 months after birth werebled and the concentration of human antibody κ chain in the sera wasdetermined by ELISA in the same manner as in Example 20.

[0403] The results are shown in Table 18 together with the data obtainedin Example 42 on the retention of chromosome. It was confirmed that thehuman antibody κ chain gene can function in the offspring micereproduced from the chimeric mice. TABLE 18 Concentration of HumanAntibody κ Chain in Offspring Mice (ELISA) Presence of Number of humanchromosome #2 Mother mouse mouse fragments Ig κ (mg/l) K2-1F #1 − 0.58K2-1F #2 + 84.1 K2-1F #3 + 12.8 K2-1F #4 + 15.1 K2-1F #5 − 0.52 K2-1F #6− 0.58 K2-1F #7 − 1.30 K2-1F #8 − 0.90 K2-1F #9 − 0.56 K2-1F #10  + 28.8K2-4F #1 − <0.04 K2-4F #2 + 23.3 K2-4F #3 + 11.8 K2-4F #4 − 0.08 K2-4F#5 − 0.06

EXAMPLE 45

[0404] Analysis of Spleen Cells from the Human Chromosome #14 PartialFragment Transferred Chimeric Mice

[0405] Flow cytometry analysis was accordance with the method describedin “New Biochemical Experiment Lecture 12, molecular immunologyI-Immunocells•Cytokines-”, edited by the Japanese Biochemical Society,1989, published by Tokyo Kagaku Dojin; “Cell Technology Separated Volume8, New Cell Technology Experiment Protocol”, edited by the University ofTokyo, Medical Science Institute, Anti-cancer Laboratory, 1991,published by Shujunsha; and A. Doyle and J. B. Griffiths, “Cell & TissueCulture: Laboratory Procedures”, published by John Wiley & Sons Ltd.,1996. The spleen was removed from the chimeric mouse KPG06 (derived fromthe PG16 clone, 30% chimerism) from Example 19 of six months after birthand treated with an aqueous solution of ammonium chloride. The spleencells were stained with fluorescein isothiocyanate (FITC)-labeledanti-mouse CD45R (B220) antibody (Pharmingen, 01124A) in PBS containing1% rat serum. After being washed, the cells were reacted with 0.1 μg ofbiotin-labeled anti-human IgM antibody (Pharmingen, 08072D) or a controlbiotin-labeled anti-human λ chain antibody (Pharmingen, 08152D) in PBScontaining 5% mouse serum and stained with 0.1 μg ofstreptoavidin-phycoerythrin (Pharmingen, 13025D), followed by analysiswith a flowcytometer (Becton Dickinson Immunocytometry Systems,FACSort). An ICR mouse retaining no human chromosome was used as acontrol for analysis by the same method. The results are shown in FIG.22. In FIG. 22, the human IgM is plotted on the horizontal axis and theCD45R (B220) is plotted on the vertical axis. A population of cellspositive to both B cell marker CD45R (FITC) and human IgM (PE) increasedby 4%, indicating that cells expressing human antibody μ chain on thecell surfaces were present in the chimeric mice.

EXAMPLE 46

[0406] Cloning and Sequencing of Variable Regions of Human AntibodyGenes from cDNA Derived from the Spleen of the Chimeric Mice ExpressingHuman Antibody Heavy Chain, κ and λ Chains, Respectively

[0407] In the same manner as in Example 5, cDNAs were synthesized fromRNAs extracted from the spleens of the chimeric mice K15A (derived fromthe 1-4 clone, prepared by the method described in Example 10), K2-8prepared in Example 13 and KPG22-2 prepared in Example 31, all of whichwere confirmed to express human antibody heavy chain, κ and λ chains inExamples 29, 23 and 32, respectively. PCR was performed using therespective cDNAs and the following primers to amplify the variableregions of respective human antibody. cDNA derived from the spleen of anon-chimeric mouse ICR was used as a negative control. Those primers setforth below without indication of reference literature were designed onthe basis of the nucleotide sequences obtained from data bases such asGenebank and the like.

[0408] K15A (Heavy Chain)

[0409] For constant region: HIGMEX1-2: 5′-CCAAGCTTCAGGAGAAAGTGATGGAGTC(SEQ ID NO:53) HIGMEX1-1: 5′-CCAAGCTTAGGCAGCCAACGGCCACGCT (used in 2ndPCR of (SEQ ID NO:54) VH3BACK)

[0410] For variable region:

[0411] VH1/5BACK (59° C., 35 cycles, Marks et al., Eur. J. Immnol., 21,985-, 1991),

[0412] VH4BACK (59° C., 35 cycles, Marks et al., supra), and

[0413] VH3BACK (1st PCR:59° C., 35 cycles; 2nd PCR:59° C. , 35 cycles,Marks et al., supra)

[0414] K2-8 (Light Chain κ)

[0415] For constant region:

[0416] KC2H: 5′-CCAAGCTTCAGAGGCAGTTCCAGATTTC (SEQ ID NO: 55)

[0417] For variable region:

[0418] Vk1/4BACK (55° C., 40 cycles, Marks et al., Eur. J. Immnol., 21,985-, 1991),

[0419] Vk2BACK (55° C., 40 cycles, Marks et al., supra), and

[0420] Vk3BACK (55° C., 40 cycles, Marks et al., supra)

[0421] KPG22-2 (Light Chain λ)

[0422] For constant region:

[0423] Cλ MIX (a mixture of the following three kinds of primers at anequal molar ratio) IGL1-CR: 5′-GGGAATTCGGGTAGAAGTCACTGATCAG (SEQ IDNO:56) IGL2-CR: 5′-GGGAATTCGGGTAGAAGTCACTTATGAG (SEQ ID NO:57) IGL7-CR:5′-GGGAATTCGGGTAGAAGTCACTTACGAG (SEQ ID NO:58)

[0424] For variable region:

[0425] Vλ 1LEA1 (55° C., 40 cycles, Williams et al., Eur. J. Immunol.,23, 1456-, 1993),

[0426] Vλ 2MIX (55° C., 40 cycles, a mixture of Vλ 2 LEAL and Vλ 2 JLEAD(Williams et al. (supra)) at an equal molar ratio)

[0427] Vλ 3MIX (55° C., 40 cycles, a mixture of Vλ 3LEA1, Vλ 3JLEAD andVλ 3BACK4, which were reported in Williams et al. (supra) at an equalmolar ratio.

[0428] The PCR was performed with combinations of the primers forconstant regions with those for variable regions (3 primer pairs eachfor heavy chain, κ and λ chains) at 94° C. for 15 seconds, at theannealing temperatures shown with respect to the respective primers forvariable region for 15 seconds, at 72° C. for 20 seconds in the cyclenumbers shown with respect to the respective primers for variable regionusing a Thermal Cycler 9600 (Perkin-Elmer Corp.). In the second run ofPCR using VH3BACK, the amplification products of the first run of PCRwere amplified again with a combination of the two primers H1GMEX1-1 andVH3BACK. All of the amplification products were electrophoresed on a1.5% agarose gel and stained with ethiduim bromide for detection. As aresult, the amplification products having expected lengths (heavy chain,about 470 bp; light chain κ, about 400 bp; and light chain λ, about 510bp) were detected in all of the combinations. In the negative control,specific amplification product was not detected at the same position inany of the combinations. The obtained amplification products wereextracted from the agarose gel using prep.A.gene (Bio-Rad Laboratories,Inc.), treated with restriction enzymes (heavy chain, HindIII and PstI;light chain κ, HindIII and PvuII; and light chain λ, HindIII and EcoRI),and cloned into pUC119 (TAKARA SHUZO CO., LTD.) at the sites ofHindIII/PstI (heavy chain), HindIII/HincII (κ chain) and HindIII/EcORI(λ chain). The nucleotide sequences of the products that were amplifiedwith the following primers and which were cloned into the plasmids weredetermined with a Fluorescence Autosequencer (Applied Biosystems Inc.).

[0429] HIGMEX1-2×VH1/5BACK: 10 clones

[0430] HIGMEX1-2×VH4BACK: 8 clones

[0431] HIGMEX1-2 (2nd PCR, HIGMEX1-1)×VH3BACK: 5 clones

[0432] KC2H×V κ 1/4BACK: 6 clones

[0433] KC2H×V κ 2BACK: 7 clones

[0434] KC2H×V κ 3BACK: 4 clones

[0435] Cλ MIX×Vλ 1LEA1: 5 clones

[0436] Cλ MIX×Vλ 2MIX: 6 clones

[0437] Cλ MIX×Vλ 3MIX: 5 clones

[0438] The obtained nucleotide sequences were analyzed with DNASIS(Hitachi Software Engineering Co., Ltd.). The results show that all ofthe sequences were derived from human and that they were functionalsequences which did not contain a termination codon at any site betweenan initiation codon and a constant region: this was true with all of theκ and λ chains and with 21 out of a total of 23 heavy chains. When thesame sequences were removed from the determined sequences, uniquevariable region sequences were identified as follows: 17 heavy chains,11 κ chains, and 12 λ chains.

EXAMPLE 47

[0439] Analysis of the Nucleotide Sequences of Variable Region of HumanAntibody Genes from cDNA Derived from the Spleen of the Chimeric MouseExpressing Human Antibody Heavy Chain, κ and λ Chains, Respectively

[0440] The nucleotide sequences determined in Example 46 (heavy chain,17 clones; κ chain, 11 clones; and λ chain, 12 clones) were analyzed inthe following points.

[0441] 1. Identification of known germ line V gene segments used in therespective variable regions

[0442] 2. Identification of known germ line J gene segments used in therespective variable regions

[0443] 3. Identification of known germ line D gene segments used in theheavy chain variable regions

[0444] 4. Identification of the addition of N region in the heavy chainvariable regions on the basis of the results of 1, 2 and 3

[0445] 5. Determination of the amino acid sequences deduced from thenucleotide sequences of the respective variable regions

[0446] The results are shown in Table 19. For the identification inpoints of 1 and 2, search for homology with germ line V and J segmentsregistered in Genbank and the like was conducted with DNASIS. The VHsegments, V κ segments and Vλ segments are shown in Table 19 togetherwith the family names of the respective V fragments in accordance withthe conventions described in Cook et al., Nature genetics, 7, 162-, 1994(VH fragments), Klein et al., Eur. J. Immunol, 23, 3248-, 1993 (V κfragments) and Williams et al. (supra) (Vλ0 fragments), respectively.For the identification in point 3, search for homology with germ line Dfragments reported in Ichihara et al., The EMBO J., 7, 13, 4141-, 1988was conducted with DNASIS. Assignment was based on at least 8 bpidentity and the results are shown in Table 19. DN1★ is believed to bethe new DN family segment reported in Green et al., Nature Genetics, 7,13-, 1994. For the identification in point 4, the nucleotide sequenceswhich did not appear in any germline sequences were determined to be Nregions on the basis of the results for 1(V), 2(J) and 3 (D). As aresult, N region was observed in 11 of the 13 sequences in which Dsegment was identified and its average length was 8.7 bp. For thedetermination in point 5, the respective sequences were converted byDNASIS to amino acid sequences which were expressed with one lettersymbols. In Table 19, only CDR3 region is shown. At the right side ofTable 19, the names of the primers used in cloning of the respectivevariable regions and the names of clones are shown. TABLE 19 V family Vsegment CDR3 J (D) V primer Clone K15A VH1 VH1-8 VRSSSWYEYYYYGMDV J6(DN1) VH4BACK H4-10 VH1-18 GGITMVRGLIITDWYFDL J2 (DXP′1) VH1/5BACK H1-7VH1-24 APYSGRFDY J4 (DK1) VH1/5BACK H1-6 VH1-46 ERYYGSGSYQDYYYYYGMDV J6(DXP′1) VH1/5BACK H1-2 VH1-46 GGYSGYEDYYYYGMDV J6 (DK1) VH1/5BACK H1-10VH2 VH2-5 SYFDWPDFDY J4 (DXP1) VH4BACK H4-14 VH3 VH3-21EGCSGGSCLPGYYYYGMDV J6 (DLR2) VH1/5BACK H1-4 VH3-23 AHGDPYFDY J4VH1/5BACK H1-3 VH3-23 DADAFDI J3 VH1/5BACK H1-8 VH3-23 SGWDY J4 (DN1*)VH3BACK H3-3 VH3-23 TGFDL J2 VH4BACK H4-4 VH3-33 EGGYGSVGDYYYYGMDV J6(DXP′1) VH1/5BACK H1-9 VH3-33 GGYSYGYDYYYYGMDV J6 (DXP′1) VH3BACK H3-5VH3-33 GYSSGWYDY J4 (DN1*) VH4BACK H4-9 VH4 VH4-34 RYSSGWYYFDY J4 (DN1*)VH4BACK H4-15 VH4-59 GRIAVASFDY J4 (DN1*) VH4BACK H4-2 VH4-59 GSGSYFHFDYJ4 VH4BACK H4-6 K2-8 V κ 1 O18-8 QQHDNLPFT J3 V κ 1BACK K1-1 O18-8QQYDNLPIT J5 V κ 1BACK K1-3 O18-8 QQHDNLPFA J3 V κ 2BACK K2-2 L1QQYNSYPLT J4 V κ 1BACK K1-6 V κ 2 A17 MQGTHLLT J4 V κ 2BACK K2-1 A17MQGTHWIT J5 V κ 2BACK K2-5 V κ 3 A27 QQYGSSPTWT J1 V κ 3BACK K3-1 A27QQYGSSPFT J3 V κ 3BACK K3-4 A27 QQYGSSPLWT J1 V κ 3BACK K3-5 A27QQYGSSPPWT J1 V κ 3BACK K3-6 V κ 6 A26-10 HQSSSLPQT J1 V κ 2BACK K2-4KPG22-2 V λ 1 DPL3 AAWDDSLDVV JC3 V λ 1LEA1 L1-3 DPL5 GTWDSSLSAGV JC2 Vλ 1LEA1 L1-4 DPL5 GTWDSSLSAGVV JC3 V λ 1LEA1 L1-6 DPL5 GTWDSSLSAVV JC2 Vλ 1LEA1 L1-9 DPL8 QSYDSSLSGVV JC3 V λ 1LEA1 L1-8 V λ 2 DPL10 CSYAGSSTLVJC2 V λ 2MIX L2-4 DPL11 SSYTSSSTVV JC2 V λ 2MIX L2-1 DPL11 SSYTSSSTLVJC2 V λ 2MIX L2-3 DPL11 CSYTSSSTFV JC2 V λ 2MIX L2-7 DPL12 SSYAGSNNLVJC3 V λ 2MIX L2-5 DPL12 SSYAGSNNFVV JC3 V λ 2MIX L2-6 V λ 3 DPL16NSRDSSGNLV JC2 V λ 3MIX L3-1

EXAMPLE 48

[0447] Preparation of a Targeting Vector for Knocking Out Antibody Genes(Heavy-Chain and Light-Chain κ Genes) in TT2 (or TT2F) ES Cells

[0448] It becomes possible to transfer a human chromosome #14 fragmentmarked with a G418 resistance gene (Example 9) and human chromosome #2(Example 18) or #22 (Example 35) marked with a puromycin resistance geneinto TT2 (or TT2F) cells in which mouse antibody genes (heavy-chain,light-chain κ) are disrupted. Those chimeric mice which are producedfrom these human chromosomes #14+#2 or #14+#22-transferred, mouseantibody genes (heavy-chain, light-chain κ)-disrupted TT2 (or TT2F) EScells according to the method of Example 19 (heavy-chain+κ chain) orExample 36 (heavy-chain+λ chain) are expected to produce antibodies bothheavy- and light-chains of which are mainly derived from humans. Theabbreviations of the restriction enzymes, etc. appearing in FIGS. 23-27are as follows.

[0449] Restriction enzymes: Kp: KpnI, B: BamHI, RI: EcoRI, RV: EcoRV, N:NotI, SII: ScaII, Sca: ScaI, Sfi: SfiI, Sm: SmaI, X: XhoI, SI: SalI,dKp: deletion of KpnI, (X): XhoI restriction site from λ vector

[0450] Dotted portion: pBluescript SKII(+) plasmid DNA

[0451]

: LoxP sequence

[0452] 1. Preparation of Plasmid LoxP-pstNEO in Which LoxP Sequence isInserted at Both the Ends of a G418 Resistance Gene

[0453] For the deletion of a G418 resistance gene after knocking out anantibody gene of TT2 or (TT2F) cells, it is necessary to insert LoxPsequence (Sauer et al., Proc. Natl. Acad. Sci. USA, 85, 5166-, 1988)which is the recognition sequence of Cre recombinase (Sauer et al.,supra) at both the ends of the G418 resistance gene (Example 1) in thesame direction. Briefly, pstNEO gene was cut out from pSTneoB plasmidDNA (Example 1) with restriction enzyme XhoI. The DNA fragment waspurified by agarose gel electrophoresis and then blunted with T4-DNApolymerase (Blunting End Kit from Takara Shuzo). LoxPsequence-containing plasmid DNA pBS246 (Plasmid pBS246, loxP2 CassetteVector, U.S. Pat. No 4,959,317) was purchased from GIBCO BRL. XhoIlinker DNAs were inserted into the EcoRI and SpeI restriction sites ofthis plasmid. The pstNEO DNA fragment described above was inserted intothe EcoRV restriction site of the thus modified pBS246 to give plasmidLoxP-pstNEO (FIG. 23).

[0454] 2. Isolation of Genomic DNA Clones Containing C57BL/6-DerivedAntibody Heavy-Chain C μ (IgM Constant Region) or Light-Chain Jκ-Cκ (IgκJoint Region and Constant Region)

[0455] Since TT2 (or TT2F) cells were derived from F1 mice betweenC56BL/6 mice and CBA mice, the inventors have decided to prepare vectorsfor antibody gene knockout using genomic DNA clones derived from aC57BL/6 mouse. As a genomic DNA library, an adult C57BL/6N maleliver-derived λ DNA library from Clontech was used. As a probe forscreening, the following synthetic DNA sequences (60 mers) were used.Heavy-chain Cμ probe: 5′-ACC TTC ATC GTC CTC TTC CTC CTG AGC CTC TTC(SEQ ID NO:59) TAC AGC ACC ACC GTC ACC CTG TTC AAG-3′ Light-chainκ probe: 5′-TGA TGC TGC ACC AAC TGT ATC CAT CTT CCC ACC ATC (SEQ IDNO:60) CAG TGA GCA GTT AAC ATC TGG AGG-3′

[0456] The λ clones were isolated and analyzed to subclone those DNAfragments containing heavy-chain C μ or light-chain Jκ-Cκ into plasmidpBluescript SKII(+) (Stratagene) (heavy-chain Cμ: FIG. 24; light-chainJκ-Cκ: FIG. 25). These DNA fragments were used to prepare targetingvectors for disrupting mouse antibody genes in TT2 (or TT2F) cells asdescribed below.

[0457] 3. Preparation of a Vector Plasmid for Disrupting a MouseAntibody Heavy-Chain Gene

[0458] In the Cμ-encoding region in the genomic DNA fragment containinga mouse antibody heavy-chain constant region which was prepared in 2above, a DNA fragment containing the 2nd to 4th exons (BamHI-XhoI) wasreplaced with the LoxP-pstNEO gene prepared in 1 above (FIG. 26). Thedirection of transcription of pstNEO was opposite to the direction oftranscription of the antibody gene. This plasmid DNA was amplified usingE. coli JM109 and purified by cesium chloride equilibrium centrifugation(“Introduction to Cell Technology Experimental operations”, published byKodansha, 1992). The purified plasmid DNA was cleaved at one site withrestriction enzyme SacII and used for transfection of TT2 (or TT2F) EScells. As a probe for Southern blot analysis of transformant genomic DNAto detect from transformant TT2 (or TT2F) ES cells those clones in whichhomologous recombination has taken place in the antibody heavy-chainportion with the targeting vector, a DNA fragment (about 500 bp) of theswitch region located upstream of C μ-encoding region. This DNA fragmentwas obtained by amplifying 129 mouse genomic DNAs by PCR under thefollowing conditions. Sense primer: 5′-CTG GGG TGA GCC GGA TGT TTT G-3′(SEQ ID NO: 61) Antisense primer: 5′-CCA ACC CAG CTC AGC CCA GTT C-3′(SEQ ID NO: 62)

[0459] Template DNA: 1 μg of EcoRI-digested 129 mouse genomic DNAs Thereaction buffer, deoxynucleotide mix and Taq DNA polymerase used werefrom Takara Shuzo.

[0460] Reaction conditions: 94° C., 3 min, 1 cycle→94° C., 1 min; 55°C., 2 min; 72° C., 2 min; 3 cycles→94° C., 45 sec; 55° C., 1 min; 72°C., 1 min; 36 cycles

[0461] After it was confirmed that amplified DNA fragment can be cleavedat one site with restriction enzyme HindIII as indicated in the Genbankdatabase, this DNA fragment was subcloned into the EcoRV restrictionsite of plasmid pBluescript. This plasmid DNA (S8) was digested withrestriction enzymes BamHI and XhoI. A PCR fragment (about 550 bp) waspurified by agarose gel electrophoresis to give a probe. Genomic DNAfrom those TT2 (or TT2F) ES cells transformed with the targeting vectoris digested with restriction enzymes EcoRI and XhoI, and separated byagarose gel electrophoresis. Then, Southern blotting is performed usingthe above probe.

[0462] 4. Preparation of a Vector for Disrupting the Mouse AntibodyLight-Chain κ Gene

[0463] The genomic DNA fragment prepared in 2 above contains the Jregion and constant region of mouse antibody light-chain κ. A DNAfragment (EcoRI-SacII) containing the J region (J1-J5) was replaced withthe LoxP-pstNEO gene prepared in 1 above (FIG. 27). The direction oftranscription of pstNEO was the same as that of the antibody gene. Thisplasmid DNA was amplified using E. coli JM109 and purified by cesiumchloride equilibrium centrifugation. The purified plasmid DNA wascleaved at one site with restriction enzyme KpnI and used fortransfection of TT2 (or TT2F) ES cells. As a probe for Southern blotanalysis of transformant genomic DNA to detect from transformant TT2 (orTT2F) ES cells those clones in which homologous recombination has takenplace in the antibody heavy-chain portion with the targeting vector aDNA fragment at the 3′ of the light-chain Jκ-Cκ genomic DNA fragment(see FIG. 25) (XhoI-EcoRI; about 1.4 kbp) was used. Genomic DNA fromthose TT2 (or TT2F) ES cells transformed with the targeting vector isdigested with restriction enzymes EcoRI and NotI, and separated byagarose gel electrophoresis. Then, Southern blotting is performed usingthe above probe.

EXAMPLE 49

[0464] Production of a Mouse ES Cell Antibody Heavy-Chain Gene-DisruptedClone

[0465] In order to obtain a recombinant in which an antibody heavy-chaingene has been disrupted by homologous recombination (hereinafter,referred to as an “antibody heavy-chain homologous recombinant”), theantibody heavy-chain targeting vector prepared in Section 3, Example 48was linearized with restriction enzyme-SacII (Takara Shuzo), andtransferred into mouse TT2F ES cells according to the method describedby Shinichi Aizawa, “Biomanual Series 8, Gene Targeting”, published byYodosha, 1995. The TT2F cells were treated with trypsin and suspended inHBS at a concentration of 2.5×10⁷ cells/ml. To the cell suspension, 5 μgof DNA was added. Then, electroporation was performed with a gene pulser(Bio-Rad Laboratories, Inc.; resistor unit not connected). A voltage of250 V was applied at a capacitance of 960 μF using an electroporationcell of 4 mm in length at room temperature. The electroporated cellswere suspended in 20 ml of an ES medium and inoculated into two tissueculture plastic plates (Corning) of 100 mm into which feeder cells wereseeded preliminarily. Similarly, experiments using 10 and 15 μg of DNAwere also conducted. After one day, the medium was replaced with amedium containing 300 μg/ml of G418 (GENETICIN; Sigma). Seven to ninedays thereafter, a total of 176 colonies formed were picked up. Eachcolony was grown up to confluence in a 12-well plate, and then fourfifths of the culture was suspended in 0.2 ml of a preservation medium[ES medium+10% DMSO (Sigma)] and stored frozen at −80° C. The remainingone fifth was inoculated into a 12-well gelatin coated plate andcultured for 2 days. Then, genomic DNA was obtained by the methoddescribed in Example 2. These genomic DNAs from G418 resistant TT2Fcells were digested with restriction enzymes EcoRI and XhoI (TakaraShuzo) and separated by agarose gel electrophoresis. Then, Southernblotting was performed to detect homologous recombinants with the probedescribed in Section 3, Example 48. As a result, 3 clones out of the176. clones were homologous recombinants. The results of Southern blotanalysis of wild-type TT2F cells and homologous recombinants #131 and#141 are shown in the left-side three lanes in FIG. 28. In wild-typeTT2F cells, two bands (a and b) are detected which were obtained by theEcoRI and XhoI digestion. In the homologous recombinants, it is expectedthat one of these bands disappears and that a new band (c) will appearat the lower part of the lane. Actually, band (a) has disappeared in#131 and #141 in FIG. 28 and a new band (c) has appeared. The size ofDNA is shown at the left side of the Figure. These results show that oneallele of an antibody heavy-chain gene in these recombinant clones hasbeen disrupted by homologous recombination.

EXAMPLE 50

[0466] Production of Chimeric Mice from Antibody Heavy-Chain HomologousRecombinant ES Cells

[0467] The cells in a frozen stock-of the antibody heavy-chainhomologous recombinant TT2F cell clone #131 from Example 49 were thawed,started to culture and injected into 8-cell stage embryos obtained bymating a male and a female mouse of ICR or MCH(ICR) (CREA JAPAN, INC.);the injection rate was 10-12 cells per embryo. After the embryos werecultured overnight in the medium for ES cells (see Example 9) to developinto blastocysts, about ten of the TT2F cell-injected embryos weretransplanted to each side of the uterus of a foster mother ICR mouse(CREA JAPAN, INC.; 2.5 days after pseudopregnant treatment). As a resultof transplantation of a total of 94 injected embryos, 22 offspring micewere born. Chimerism in the offsprings can be determined by the extentof TT2F cell-derived agouti coat color (dark brown) in the host embryo(ICR)-derived albino coat color (white). Out of the 22 offsprings, 18mice were recognized to have partial agouti coat color, indicating thecontribution of the ES cells. Out of the 18 mice, 16 mice were femalechimeric mice in which more than 80% of their coat color was agouti(i.e. ES cell-derived). From these results, it was confirmed that theantibody heavy-chain homologous recombinant ES cell clone #131 retainsthe ability to produce chimera. Since a large number of the resultantchimeric mice are female mice exhibiting extremely high contribution, itis very likely that the ES cells have differentiated into functionalgerm cells (oocytes). Two female chimeric mice exhibiting 100%contribution were mated with MCH(ICR) male mice. As a result, all of theoffspring mice exhibited agouti coat color. These offsprings are derivedfrom #131 (see Example 42), and thus it is considered that a disruptedantibody heavy-chain allele was transmitted to them at a rate of 50%.

EXAMPLE 51

[0468] Production of a Double Knockout Clone from the AntibodyHeavy-Chain Homologous Recombinant

[0469] It has been reported that a clone in which both alleles aredisrupted can be obtained by disrupting one allele by insertion of aG418 resistance gene, culturing an ES cell clone in a medium with anincreased G418 concentration and screening the resultant highconcentration G418 resistant clones (Shinichi Aizawa, “Biomanual Series8, Gene Targeting”, published by Yodosha, 1995). Based on thistechnique, the inventors have conducted the following experiments inorder to obtain both alleles-disrupted clones from the TT2F antibodyheavy-chain homologous recombinants #131 and #141. First, in order todetermine the lethal concentration of G418 for both #131 and #141clones, each clone was inoculated into ten 35 mm plates at a rate ofabout 100 cells per plate (in this Example, G418 resistant primaryculture cells which were not treated with mitomycin were used as feedercells)(see Example 9). The cells were cultured in an ES mediumcontaining 0, 0.5, 1, 2, 3, 5, 8, 10, 15 and 20 mg/ml of G418(GENETICIN, Sigma) for 10 days. As a result, definite colonies wereobserved at a concentration of up to 3 mg/ml, but no colony formationwas observed at 5 mg/ml. Based on these results, the minimum lethalconcentration was decided to be 5 mg/ml. Then, high concentration G418resistant clones were selected at concentrations of 4, 5, 6, 7 and 8mg/ml. For each of #131 and #141, cells were inoculated into ten 100 mmplates at a rate of about 10⁶ cells per plate and cultured in an ESmedium containing G418 at each of the concentrations described above (5grades; two plates for each concentration). Twelve days after the startof culture, definite colonies (#131: 12 clones; #141: 10 clones) werepicked up from plates of 7 mg/ml and 8 mg/ml in G418 concentration.These clones were stored frozen and genomic DNA was prepared by the sameprocedures as in Example 49. The genomic DNAs from these highconcentration G418 resistant clones were digested with restrictionenzymes EcoRI and XhoI (Takara Shuzo) and separated by agarose gelelectrophoresis. Then, Southern blotting was performed to detect withthe probe from Section 3, Example 48 those clones in which both alleleshave been disrupted. As a result, one clone derived from #131 (#131-3)was found to be both alleles-distrupted clone. The results of Southernblot analysis of 6 clones derived from #131 are shown in FIG. 28. Inwild-type TT2F cells, two wild-type bands (a, b) are detected after theEcoRI and XhoI digestion. In one allele homologous recombinants (#131,#141), the upper band (a) has disappeared and a new band (c) hasappeared (Example 49). Furthermore, it is expected that due to thedisruption of both alleles, another wild-type band (b) disappears andthat the disruption-type band (c) remains alone. In FIG. 28, this bandpattern is observed in clone No. 3 (#131-3). This demonstrates that bothalleles of an antibody heavy-chain gene have been disrupted in thisclone.

EXAMPLE 52

[0470] Removal of a G418 Resistance Marker Gene from the AntibodyHeavy-Chain-Deficient Homozygote TT2F Clone

[0471] The G418 resistance marker gene in the antibody heavy-chain bothalleles-disrupted clone (high concentration G418 resistant clone #131-3)from Example 51 was removed by the following procedures. An expressionvector, pBS185 (BRL), containing Cre recombinase gene which causes asite-specific recombination between the two LoxP sequences inserted atboth the ends of the G418 resistance gene was transferred into #131-3clone according to the methods described in Shinichi Aizawa, “BiomanualSeries 8, Gene Targeting”, published by Yodosha, 1995 and Seiji Takatsuet al., “Experimental Medicine (extra number): Basic Technologies inImmunological Researches”, p. 255-, published by Yodosha, 1995).Briefly, #131-3 cells were treated with trypsin and suspended in HBS togive a concentration of 2.5×10⁷ cells/ml. To the cell suspension, 30 μgof pBS185 DNA was added. Then, electroporation was performed with a genepulser (Bio-Rad Laboratories, Inc.; resistor unit not connected). Avoltage of 250 V was applied at a capacitance of 960 μF using anelectroporation cell of 4 mm in length (see Example 1). Theelectroporated cells were suspended in 5 ml of an ES medium andinoculated into a tissue culture plastic plate (Corning) of 60 mm inwhich feeder cells were seeded preliminarily. After two days, the cellswere treated with trypsin and reinoculated into three 100 mm plates(preliminarily seeded with feeder cells) such that the three plates have100, 200 and 300 cells, respectively. A similar experiment was alsoconducted under the same conditions except that the setting of the genepulser was changed (resistor unit connected; resistance value infinite).After seven days, a total of 96 colonies formed were picked up andtreated with trypsin. Then, the colonies were divided into two groups;one was inoculated into a 48-well plate preliminarily seeded with feedercells and the other was inoculated into a 48-well plate coated withgelatin alone. The latter was cultured in a medium containing 300 μg/mlof G418 (GENETICIN, Sigma) for three days. Then, G418 resistance wasjudged from the survival ratio. As a result, 6 clones died in thepresence of G418. These G418 sensitive clones were grown to confluencein 35 mm plates, and four fifths of the resultant culture was suspendedin 0.5 ml of a preservation medium [ES medium+10% DMSO (Sigma)] andstored frozen at −80° C. The remaining one fifth was inoculated into a12-well gelatin coated plate and cultured for two days. Thereafter,genomic DNA was prepared by the same procedures as in Example 2. Thesegenomic DNAs from G418 sensitive TT2F clones were digested withrestriction enzyme EcoRI (Takara Shuzo) and separated by agarose gelelectrophoresis. Then, Southern blotting was performed to confirm theremoval of the G418 resistance gene using a 3.2 kb XhoI fragment (probeA) from G418 resistance gene-containing pSTneoB. As a result, bandsobserved in #131-3 clone which hybridize with Probe A were not detectedat all in the sensitive clones. From these results, it was confirmedthat the G418 resistance marker gene had been surely removed in the G418sensitive clones obtained. Additionally, as a result of Southern blotanalysis performed in the same manner using Probe B obtained bydigesting pBS185 DNA with EcoRI, no specific band which hybridizes withProbe B was detected in these G418 sensitive clones. Thus, it isbelieved that Cre recombinase-containing pBS185 is not inserted into thechromosomes of the sensitive clones. In other words, these sensitiveclones can be transformed with the vector for knocking out an antibodylight-chain (vector having a loxP sequence at both the ends of a G418resistance gene) described in Section 4, Example 48.

EXAMPLE 53

[0472] Transfer of Human Chromosome #14 (Containing Antibody Heavy-ChainGene) into the Antibody Heavy-Chain-Deficient ES Cell Clone

[0473] Human chromosome #14 (containing an antibody heavy-chain gene)marked with a G418 resistance gene is transferred by microcell fusion asdescribed in Example 9 into the mouse ES cell clone (from TT2F, G418sensitive) obtained in Example 52 which is deficient in an endogenousantibody heavy-chain. In the resultant G418 resistant clone, theretention of human chromosome #14 (fragment) containing a human antibodyheavy-chain gene is confirmed by PCR analysis or the like (see Example9).

EXAMPLE 54

[0474] Transfer of Human Chromosome #2 Fragment or Human Chromosome #22Into the Antibody Heavy-Chain-Deficient ES Cell Clone Retaining HumanChromosome #14 (Fragment)

[0475] A human chromosome #2 fragment (containing the antibodyheavy-chain κ gene) or human chromosome #22 (containing the antibodyheavy-chain λ gene) marked with a puromycin resistance gene istransferred into the antibody heavy-chain-deficient mouse ES cell cloneretaining a human chromosome #14 partial fragment (G418 resistant) fromExample 53 by microcell fusion as described in Examples 18 and 35. Inthe resultant puromycin and G418 double drug-resistant clone, theretention of the human chromosome #14 (fragment) and human chromosome #2fragment or #22 (fragment) is confirmed by PCR analysis or the like (seeExamples 18 and 35).

EXAMPLE 55

[0476] Production of Chimeric Mice from the Endogenous AntibodyHeavy-Chain-Deficient Mouse ES Cells Retaining Human Chromosome #14(Fragment) Containing a Human Antibody Heavy-Chain Gene

[0477] Chimeric mice from the endogenous antibody heavy-chaingene-deficient mouse ES cell clone obtained in Example 53 retaininghuman chromosome #14 (fragment) containing a human antibody heavy-chaingene are produced by the same procedures as in Example 10. In theresultant chimeric mice, a human antibody heavy-chain produced in the EScell clone-derived B cells is detected by the method described inExample 14. Since antibody heavy-chain genes functional in the ES cellclone-derived B cells are only the human-derived gene on the transferredchromosome, many of the ES cell clone-derived B cells produce humanantibody heavy-chain.

EXAMPLE 56

[0478] Production of Chimeric Mice from the Endogenous AntibodyHeavy-Chain-Deficient Mouse ES Cells Retaining Human Chromosomes #14+#2(Fragments) or #14+#22 (Fragments)

[0479] Chimeric mice are produced by the same procedures as in Examples19, 36, etc from the endogenous antibody heavy-chain gene-deficientmouse ES cell clone retaining human chromosomes #14+#2 (fragments) or#14+#22 (fragments) obtained in Example 54. In the resultant chimericmice, human antibody heavy-chain and light-chain κ or γ are detected inthe ES cell clone-derived B cells according to the method described inExamples 14, 23 and 32. As in Example 55, antibody heavy-chain genesfunctional in the ES cell clone-derived B cells are only thehuman-derived gene on the transferred chromosome. Thus, many of the EScell clone-derived B cells produce human heavy-chains. Furthermore,complete human antibody molecules both heavy and light-chains which arederived from humans are also detected by the method described inExamples 37 and 38.

EXAMPLE 57

[0480] Production of Human Antibody-Producing Hybridomas from theChimeric Mice Derived from the Endogenous Antibody Heavy-Chain-DeficientMouse ES Cells Retaining Human Chromosomes #14+#2 (Fragments) or #14+#22(Fragments)

[0481] The chimeric mice from Example 56 are immunized with an antigenof interest in the same manner as in Examples 15, 25 and 34. The spleenis isolated from each mice and the spleen cells are fused with myelomacells to produce hybridomas. After cultivation for 1-3 weeks, theculture supernatant is analyzed by ELISA. The ELISA is performed by themethod described in Examples 14, 15, 21, 24, 25, 33, 34, 37 and 38. As aresult, human antibody positive clones and clones which are humanantibody positive and specific to the antigen used in the immunizationare obtained.

EXAMPLE 58

[0482] Production of an Antibody Light-Chain Gene-Disrupted Clone fromthe Antibody Heavy-Chain-Deficient Homozygote Mouse ES Cells

[0483] A homologous recombinant, which has further disruption in anantibody light-chain gene in the antibody heavy-chain-deficienthomozygote TT2F cell clone (G418 sensitive) obtained in Example 52 isproduced by the following procedures. Briefly, the antibody light-chaintargeting vector prepared in Section 4, Example 48 is linearized withrestriction enzyme KpnI (Takara Shuzo), and transferred into the aboveTT2F cell clone (G418 sensitive) according to the method described inShinichi Aizawa, “Biomanual Series 8: Gene Targeting”, published byYodosha, 1995. After 7-9 days, colonies formed are picked up. They arestored frozen and genomic DNA is prepared in the same manner as inExample 49. Genomic DNAs from G418 resistant clones are digested withrestriction enzymes EcoRI and NotI (Takara Shuzo) and separated byagarose gel electrophoresis. Then, Southern blot analysis is performedto detect homologous recombinants with the probe described in Section 4,Example 48.

EXAMPLE 59

[0484] Production of an Double Knockout Clone from the AntibodyLight-Chain Homologous Recombinant

[0485] A clone in which both alleles of a light-chain gene are disruptedis prepared from the TT2F antibody light-chain homologous recombinant(and antibody heavy-chain-deficient homozygote) clone from Example 58 bythe procedures described below. Briefly, a high concentration G418resistant clone is prepared and stored frozen, and DNA is prepared inthe same manner as in Example 51. Genomic DNA from the highconcentration G418 resistant clone is digested with restriction enzymesEcORI and NotI (Takara Shuzo) and separated by agarose gelelectrophoresis. Then, Southern blot analysis is performed to detectthose clones in which both alleles have been disrupted, with the probefrom Section 4, Example 48.

EXAMPLE 60

[0486] Removal of the G418 Resistance Gene from the AntibodyLight-Chain-Deficient Homozygote (Antibody Heavy-Chain-DeficientHomozygote) TT2F Cell Clone

[0487] The G418 resistance marker gene in the antibody light-chain bothalleles-disrupted clone (high concentration G418 resistant clone)obtained in Example 59 is removed by the same procedures as in Example52. Briefly, an expression vector, pBS185 (BRL), containing Crerecombinase gene which causes a site-specific recombination between thetwo loxp sequences inserted at both the ends of the G418 resistance gene(Section 1, Example 48) was transferred into the above clone accordingto the method described in Example 52. The resultant G418 sensitiveclones are grown to confluence in 35 mm plates, and ⅘ of the resultantculture was suspended in 0.5 ml of a preservation medium [ES medium+10%DMSO (Sigma)] and stored frozen at −80° C. by the same procedures as inExample 52. The remaining ⅕ was inoculated into a 12-well gelatin coatedplate. After cultivation for two days, genomic DNA is prepared by themethod described in Example 2. These genomic DNAs from G418 sensitiveTT2F clones are digested with restriction enzyme EcoRI (Takara Shuzo)and separated by agarose gel electrophoresis. Then, Southern blotting isperformed to confirm the removal of the G418 resistance gene using a 3.2kb XhoI fragment from G418 resistance gene-containing pSTneoB as aprobe.

EXAMPLE 61

[0488] (1) Transfer of a Human Chromosome #14 Fragment (ContainingAntibody Heavy-Chain Gene) into the Endogenous Antibody Heavy-Chain andκ Chain-Deficient ES Cell Clone

[0489] A human chromosome #14 fragment SC20 (containing a human antibodyheavy-chain gene) was transferred by microcell fusion as described inSection 2 of Example 68 into the mouse ES cell clone HKD31 (from TT2F,G418 sensitive, puromycin sensitive) obtained in Example 78 which isdeficient in both endogenous antibody heavy-chain and X chain. Themicrocell fusion and the selection of G418 resistant clones wereperformed in the same manner as in Example 2. Eight of the resultantG418 resistant clones were subjected to PCR analysis using IgM andD14S543 primers (see Example 68). As a result, both markers weredetected in 8 out of the 7 clones analyzed. Hence, it was confirmed thatthe antibody heavy-chain and κ chain-deficient ES cell clone retains thehuman chromosome #14 fragment SC20.

[0490] (2) Production of Chimeric Mice from the Endogenous AntibodyHeavy-Chain and κ Chain Genes-Disrupted Mouse ES Cells Retaining a HumanChromosome #14 Fragment (Containing Antibody Heavy-Chain Gene)

[0491] Chimeric mice were produced by the same procedures as in Example10, etc. from the endogenous antibody heavy-chain and κ chaingenes-disrupted mouse ES cell clone HKD31-8 which was obtained inSection 1 of Example 61 and which retains a human chromosome #14fragment (containing a human antibody heavy-chain gene). As a result oftransplantation of a total of 188 injected embryos, 25 offspring micewere born. Chimerism in the offsprings can be determined by the extentof TT2 cell-derived agouti coat color (dark brown) in the host embryo(ICR)-derived albino coat color (white). Out of the 25 offsprings, 17mice were recognized to have partial agouti coat color, indicating thecontribution of the ES cells. Out of the 17 mice, three were chimericmice in which more than 95% of their coat color was (ES cell-derived)agouti.

[0492] From these results, it was confirmed that the endogenous antibodyheavy-chain and κ chain genes-disrupted mouse ES cell clone retainingthe human chromosome #14 fragment (containing a human antibodyheavy-chain gene) maintains the ability to produce chimera, that is, theability to differentiate into normal tissues of mice.

[0493] (3) Detection of Human Antibody (Having Human μ, γ or α Chain) insera of the Chimeric Mice Derived from the Endogenous AntibodyHeavy-Chain and κ Chain Genes-Disrupted Mouse ES Cells Retaining a HumanChromosome #14 Fragment (Containing Antibody Heavy-Chain Gene)

[0494] The chimeric mice produced in Section 2 of Example 61 (derivedfrom HKD31-8) were bled 12 weeks (#1) or 7 weeks (#2-4) after birth. Thehuman antibody concentration in the sera was determined by ELISA in thesame manner as in Example 14. Ninety six-well microtiter plates werecoated with PBS-diluted anti-human immunoglobulin μ chain antibody(Sigma, I6385) or anti-human immunoglobulin γ chain antibody (Sigma,I3382) or anti-human immunoglobulin α chain antibody (Pharmingen,08091D) and then a serum sample diluted with mouse serum (Sigma,M5905)-containing PBS was added. Subsequently, peroxidase-labeledanti-human immunoglobulin μ chain antibody (The Binding Site Limited,MP008) or peroxidase-labeled anti-human immunoglobulin γ chain antibody(Sigma, A0170) was added to the plates and incubated. Alternatively,biotin-labeled anti-human immunoglobulin α chain antibody (Pharmingen,08092D) was added to the plates and incubated. After the plates werewashed, an avidin-peroxidase complex (Vector, ABC Kit PK4000) was addedthereto and incubated. TMBZ (Sumitomo Bakelite, ML-1120T) was added as aperoxidase substrate and then enzyme activity was determined byabsorbance measurement at 450 nm. Purified human immunoglobulins IgM(CAPPEL, 6001-1590), IgG (Sigma, I4506) and IgA (Sigma, I2636) of knownconcentrations having μ chain, γ chain and α chain, respectively, wereused as standards for determining human antibody concentrations in thesera. These standards were diluted stepwise with mouseserum-supplemented PBS. The results are shown in Table 20. Chimeric micehaving concentrations of human antibody μ and λ chains almost as high asin normal mouse sera were confirmed. Also, chimeric mice expressinghuman α chain were confirmed. Further, human immunoglobulin γ chainsub-classes were detected in the same manner as in Example 29. As aresult, all of the four subclasses (γ1, γ2, γ3 and γ4) were detected.

[0495] These results show that a human antibody heavy-chain gene isexpressed efficiently in the chimeric mice derived from the endogenousantibody heavy-chain and κ chain genes-disrupted mouse ES cellsretaining the human chromosome #14 fragment (containing an antibodyheavy-chain gene); it was also shown that not only μ chain but also allof the γ chain subclasses and α chain were expressed therein as a resultof class switching. TABLE 20 Human Antibody Concentrations in ChimericMice (ELISA) Chimeric Chimerism Human Antibody (mg/l) Mouse % IgM IgGIgA #1 90 270 1250 0.46 #2 99 370 820 0.23 #3 99 550 1460 0.32 #4 95 3402300 0.06

[0496] (4) Acquisition of Hybridomas Producing Anti-HSA AntibodyComprising Human γ Chain from the Chimeric Mice Derived from theEndogenous Antibody Heavy-Chain and κ Chain Genes-Disrupted Mouse ESCells Retaining a Human Chromosome #14 Fragment (Containing AntibodyHeavy-Chain Gene)

[0497] Chimeric mice #3 (derived from HKD31-8; chimerism 99%) and #4(chimerism 95%) which had exhibited a high human antibody γ chainconcentration in the serum in Section 3 of Example 61 were immunized asdescribed below. Human serum albumin (HSA, Sigma, A3782) dissolved inPBS was mixed with an adjuvant (MPL+TDM Emulsion, RIBI ImmunochemResearch Inc.) to prepare an HSA solution with a concentration of 0.25mg/ml. When the above-described chimeric mice became 16-week old, 0.2 mlof this HSA solution was administered intraperitoneally twice at aninterval of 2 weeks. Two weeks thereafter, the mice were immunized withhuman serum albumin dissolved in PBS and then bled. The concentration ofanti-HSA human antibody in the sera was determined by ELISA in the samemanner as in Example 14. Briefly, ELISA plates were coated with HSA andthen peroxidase-labeled anti-human Igμ antibody (The Binding Site,MP008), anti-human Igγ antibody (Sigma, A1070) and anti-human Igαantibody (Kirkegaard & Perry Laboratories Inc., 14-10-01) were used fordetection. The results are shown FIG. 33. Hybridomas were produced usinga myeloma cell SP-2/0-Ag14 (Dainippon Pharmaceutical Co., Ltd.) by themethod described in Ando, “Monoclonal Antibody Experiment ProcedureManual”, published by Kodansha Scientific in 1991. Three days after thefinal immunization, the spleen was removed from the chimeric mice andthen cell fusion was performed using PEG in the same manner as inExample 29 to prepare hybridomas. At the same time, blood samples werecollected from the mice to quantitate human Igγ subclasses in the sera.As a result, 920 mg/l of γ 1, 520 mg/l of γ 2, 11 mg/l of γ 3 and 140mg/l of γ 4 were detected in the serum of chimeric mouse #3.

[0498] The fused cells were diluted with a medium (Sanko Pure Chemical,S Cloning Medium CM-B) containing 5% HCF (Air Brown) and HAT (DainipponPharmaceutical Co., Ltd., No. 16-808-49) or 1 mg/ml of G418 to give aconcentration of 10⁶ spleen cells/ml and then dispensed into 96-wellplates (100 μl/well), followed by cultivation. At day 8 of thecultivation, the culture supernatant was collected and screened forhuman antibody-producing hybridomas by ELISA in the same manner as inExample 14. Briefly, ELISA plates were coated with a HSA solutiondissolved in CBB buffer to give a concentration of 5μg/ml.Peroxidase-labeled anti-human immunoglobulin γ chain antibody (Sigma,A0170) and TMBZ (Sumitomo Bakelite, ML-1120T) were used for detection.An absorbance about 3 times higher than the absorbance in the negativecontrol was used as a criterion for judgement. As a result, 74 positivewells were obtained from chimeric mouse #3 and 29 positive wells fromchimeric mouse #4. Also, anti-HSA antibody having human μ chain wasscreened in HSA-solution-coated plates using peroxidase-labeledanti-human immunoglobulin μ chain antibody (Tago, #2392). Briefly, fusedcells from chimeric mouse #3 were inoculated into fifteen 96-wellplates, from which 4 plates were selected by G418 resistance. Theculture supernatants of these 4 plates were screened to obtain 5positive wells. Wells which exhibited colony formation after selectionwith HAT or 1 mg/ml of G418 were 74 wells/plate for HAT and 29wells/plate for G418. The cells of those wells which were positive forhuman γ chain-containing anti-HSA antibody and which had a relativelylarge number of cells were transferred into 46-well plates and culturedfor another 4 days. The isotype of the antibody in the culturesupernatant was determined by ELISA. ELISA was performed in HSA-coatedplates using alkali phosphatase-labeled anti-human IgG1 antibody (ZymedLabolatories, Inc., 05-3322), anti-human IgG2 antibody (ZymedLabolatories, Inc., 05-3522), anti-human IgG3 antibody (ZymedLabolatories, Inc., 05-3622) and anti-human IgG4 antibody (ZymedLabolatories, Inc., 05-3822) in the same manner as in Example 14. As aresult, 27 human IgG1 positive clones, 11 human IgG2 positive clones, 2human IgG3 positive clones and 13 human IgG4 positive clones wereobtained. Fused cells from chimeric mouse #4 were treated in the samemanner to obtain 4 positive clones with a large number of cells as humanIgG1 producing clones.

[0499] These results show that the immunization by human protein (HSA)of the chimeric mice derived from the endogenous antibody heavy-chain &light-chain-deficient mouse ES cells retaining the human chromosome #14partial fragment containing a human antibody heavy-chain gene increasesthe antibody titers of antigen specific human Igμ, γ and α to therebyenable the acquisition of hybridomas producing anti-HSA antibodycontaining μ chain and all of the human γ chain subclasses.

EXAMPLE 62

[0500] Transfer of Human Chromosome #2 (Containing Light-Chain κ Gene)Into the Endogenous Antibody Heavy-Chain and κ Chain-Deficient ES CellsRetaining a Human Chromosome #14 Fragment (Containing AntibodyHeavy-Chain Gene)

[0501] A human chromosome #2 fragment (containing antibody light-chain κgene) marked with a puromycin resistance gene was transferred into theendogenous antibody heavy-chain and κ chain-deficient mouse ES cellclone HKD31-8 obtained in Section 1 of Example 61 and which retained ahuman chromosome #14 fragment (containing an antibody heavy-chain gene).The method of transfer was by microcell fusion as described in Example18. As a result, 13 puromycin and G418 double-resistant clones wereobtained. These clones were subjected to PCR analysis (see Example 18)using IgM and D14S543 primers (see Example 68) for the chromosome #14fragment and Vκ 1 and FABP1 primers (see Example 12) for the chromosome#2 fragment. As a result, the presence of all the 4 markers wasconfirmed in 8 clones. Of these clones, KH13 clone was subjected to FISHanalysis using human chromosome-specific probes (see Examples 9 and 12).The results are shown in FIG. 34. Two independent, small chromosomefragments hybridizing to the probes were observed in KH13. These resultsshow that KH13 retains both the chromosome #14 fragment and thechromosome #2 fragment.

EXAMPLE 63

[0502] Transfer of Human Chromosome #22 (Containing Light-Chain λ Gene)Into the Endogenous Antibody Heavy-Chain and κ Chain-Deficient ES CellsRetaining a Human Chromosome #14 Fragment (Containing AntibodyHeavy-Chain Gene)

[0503] Human chromosome #22 (containing antibody light-chain λ gene)marked with a puromycin resistance gene was transferred into mouse EScell clone HKD31-8 obtained in Section 1 of Example 61 which wasdeficient in the endogenous antibody heavy-chain & κ chain and whichretained a human chromosome #14 fragment (containing an antibodyheavy-chain gene). The method of transfer was by microcell fusion asdescribed in Example 35. As a result, 12 puromycin and G418 doubledrug-resistant clones were obtained. These clones were subjected to PCRanalysis (see Example 35) using IgM and D14S543 primers for thechromosome #14 fragment and Ig λ, D22S315, D22S275, D22S278, D22S272 andD22S274 primers (see Example 2) for the chromosome #22 fragment. As aresult, the presence of all of the 8 markers was confirmed in 10 clones.Of the remaining 2 clones, LH13 clone exhibited the presence of 5markers, IgM, D14S543, IgI, D22S275 and D22S274. Thus, it is believedthat this clone contains a fragment of human chromosome #22. LH13 wasfurther subjected to FISH analysis using a human chromosome #22-specificprobe and a human chromosome #14-specific probe separately. As a result,independent chromosome fragments hybridizing to the respective probeswere observed. This indicates that this clone retains both a chromosome#14 fragment and a chromosome #2 fragment.

EXAMPLE 64

[0504] Production of Endogenous Antibody Heavy-Chain &Light-Chain-Deficient Mouse ES Cells Retaining Three Human Chromosomes,#2 (Containing Antibody Light-Chain κ Gene), #14 (Containing AntibodyHeavy-Chain Gene) and #22 (Containing Antibody λ Chain Gene), or PartialFragments Thereof

[0505] In order to obtain mouse ES cells retaining three kinds of humanchromosomes, human chromosome #2 or #22 is marked by inserting a markergene such as blasticidin resistance (Izumi et al., Exp. Cell. Res., 197:229, 1991), hygromycin resistance (Wind et al., Cell, 82:321-, 1995),etc. This marking is performed according to the method described inExamples 16 and 26. Human chromosome #22 (containing human antibodylight-chain λ gene) marked with blasticidin resistance, hygromycinresistance, etc. is transferred into the mouse ES cell clone (from TT2F,G418 resistant, puromycin resistant) obtained in Example 62 which isdeficient in endogenous antibody heavy-chain & light-chain and whichretains both human chromosome #14 (fragment) and human chromosome #2(partial fragment). The method of transfer is by the method described inExample 9. As feeder cells for culturing ES cells, appropriate cells areselected depending on the selection marker used. When a hygromycinresistance marker is used, primary culture fibroblasts obtained from atransgenic mouse strain which retains and expresses the marker (Johnsonet al., Nucleic Acids Research, vol. 23, No. 7, 1273-, 1995) are used.It is confirmed by PCR analysis, etc. (see Examples 9, 18 and 35) thatthe resultant G418, puromycin and hygromycin (or blasticidin) tripledrug-resistant clones retain the three kinds of human chromosomes(fragments) described above. In the same manner, a human chromosome #2fragment marked with a hygromycin or blasticidin resistance gene istransferred into the mouse ES cell clone (from TT2F, G418 resistant,puromycin resistant) obtained in Example 63 which is deficient inendogenous antibody heavy-chain & light-chain and which retains bothhuman chromosome #14 (fragment) and human chromosome #22 (fragment).

EXAMPLE 65

[0506] Production of Chimeric Mice from the Endogenous AntibodyHeavy-Chain & Light-Chain Genes-Disrupted Mouse ES Cells Retaining aPlurality of Human Chromosomes (Fragments) Containing Human AntibodyHeavy-Chain Gene and Light-Chain Gene, Respectively

[0507] Chimeric mice are produced by the same procedures as in Example10, etc. from the endogenous antibody heavy-chain & light-chaingenes-disrupted mouse ES cell clones that retain human chromosomes(fragments) containing human antibody genes and which were obtained inExamples 61, 62, 63 and 64. In the resultant chimeric mice, mouseantibodies produced in host embryo-derived B cells and human antibodiesproduced mainly in ES cell clone-derived B cells are detected by themethod described in Examples 14, 23 and 32. Since the antibodyheavy-chain gene and the light-chain κ gene which are both functional inthe ES cell clone-derived B cells are only human-derived genes on thetransferred chromosomes, many of the ES cell clone-derived B cellsproduce human antibody heavy-chain and light-chain κ (Lonberg et al.,Nature, 368:856-, 1994). Furthermore, complete human antibody moleculesin which both heavy- and light-chains are derived from human are alsodetected by the method described in Examples 37 and 38.

[0508] (1) Production of Chimeric Mice from the Endogenous AntibodyHeavy-Chain and κ Chain Genes-Disrupted Mouse ES Cells Retaining Both aHuman Chromosome #14 Fragment (Containing Antibody Heavy-Chain Gene) anda Human Chromosome #2 Fragment (Containing Antibody Light-Chain κ Gene)

[0509] Chimeric mice were produced by the same procedures as in Example10, etc. from mouse ES cell clone KH13 obtained in Example 62 which isdeficient in the endogenous antibody heavy-chain and κ chain genes andwhich retains both a human chromosome #14 fragment (containing anantibody heavy-chain gene) and a human chromosome #2 fragment(containing light-chain κ gene). As a result of transplantation of atotal of 176 injected embryos, 20 offspring mice were born. Chimerism inthe offsprings can be determined by the extent of TT2 cell-derivedagouti coat color (dark brown) in the host embryo (ICR)-derived albinocoat color (white). Out of the 20 offsprings, 7 mice were recognized tohave partial agouti coat color, indicating the contribution of the EScells.

[0510] From-these results, it was confirmed that the endogenous antibodyheavy-chain and κ chain genes-disrupted mouse ES cell clone retainingboth a human chromosome #14 fragment (containing an antibody heavy-chaingene) and a human chromosome #2 fragment (containing light-chain κ gene)maintains the ability to produce chimera, that is, the ability todifferentiate into normal tissues of mice.

[0511] (2) Production of Chimeric Mice from the Endogenous AntibodyHeavy-Chain and κ Chain Genes-Disrupted Mouse ES Cells Retaining Both aHuman Chromosome #14 Fragment (Containing Antibody Heave-Chain Gene) anda Human Chromosome #22 Fragment (Containing Light-Chain λ Gene)

[0512] Chimeric mice were produced by the same procedures as in Example10, etc. from mouse ES cell clone LH13 obtained in Example 63 which isdeficient in the endogenous antibody heavy-chain and κ chain genes andwhich retains both a human chromosome #14 fragment (containing anantibody heavy-chain gene) and a human chromosome #22 fragment(containing light-chain λ gene). As a result of transplantation of atotal of 114 injected embryos, 22 offspring mice were born. Chimerism inthe offsprings can be determined by the extent of TT2 cell-derivedagouti coat color (dark brown) in the host embryo (ICR)-derived albinocoat color (white). Out of the 22 offsprings, 5 mice were recognized tohave partial agouti coat color, indicating the contribution of the EScells.

[0513] From these results, it was confirmed that the endogenous antibodyheavy-chain and κ chain genes-disrupted mouse ES cell clone retainingboth a human chromosome #14 fragment (containing an antibody heavy-chaingene) and a human chromosome #22 fragment (containing light-chain λgene) maintains the ability to produce chimera, that is, the ability todifferentiate into normal tissues of mice.

[0514] (3) Detection and Quantitative Determination of Complete HumanAntibody in sera of the Chimeric Mice Derived from the EndogenousAntibody Heavy-Chain and κ Chain-Deficient Mouse ES Cells Retaining Botha Human Chromosome #2 Partial Fragment and a Human Chromosome #14Partial Fragment

[0515] The chimeric mice (derived from KH13) produced in Section 1 ofExample 65 were bled at day 40 after birth. The concentrations of humanantibody in the sera were determined by ELISA in the same manner as inExample 14. Briefly, ELISA plates were coated with PBS-dilutedanti-human immunoglobulin κ chain antibody (Kirkegaard & PerryLabolatories Inc., 01-10-10) or anti-human immunoglobulin κ chainantibody (Vector, AI-3060) and then serum samples diluted with mouseserum (Sigma, M5905)-supplemented PBS were added. Subsequently,peroxidase-labeled anti-human immunoglobulin g chain antibody (TheBinding Site Limited, MP008) or peroxidase-labeled anti-humanimmunoglobulin γ chain antibody (Sigma, A0170) was added and incubated.TMBZ (Sumitomo Bakelite, ML-1120T) was added as a peroxidase substrateand then enzyme activity was determined by absorbance measurement at 450nm. Purified human immunoglobulins IgM (Caltag, 13000) and IgG (Sigma,I4506) of known concentrations having μ chain and κ chain were used asstandards for determining human antibody concentrations in the sera bycomparison. These standards were diluted stepwise with mouseserum-supplemented PBS. The results are shown in Table 21. Chimeric micewere confirmed that had concentrations of complete human antibody morethan 10 times higher than in chimeric mice derived from ES cells whoseendogenous antibody genes were not knocked out. Also, complete humanantibody containing human_(γ) chain was confirmed in the sera of thechimeric mice.

[0516] From these results, it was confirmed that the concentration ofcomplete human antibody in which both heavy- and light-chains werederived from human increasesed in the chimeric mice derived from theendogenous antibody heavy-chain and K chain-deficient mouse ES cellsretaining both a human chromosome #14 partial fragment and a humanchromosome #22 partial fragment. TABLE 21 Concentrations of HumanAntibodies in Chimeric Mice (ELISA) ES Chimeric clone mouse Chimerism(%) IgM, κ (mg/l) IgG, κ (mg/l) KH13 CKH13-1 95 0.1 0.07 KH13 CKH13-2 850.9 0.13

[0517] (4) Detection and Quantitative Determination of Complete HumanAntibody in sera of the Chimeric Mice Derived from the EndogenousAntibody Heavy-Chain and κ Chain-Deficient Mouse ES Cells Retaining Botha Human Chromosome #14 Partial Fragment and a Human Chromosome #22Partial Fragment

[0518] The chimeric mice (derived from KH13) produced in Section 2 ofExample 65 were bled at day 49 after birth. The concentrations of humanantibody in the sera were determined by ELISA in the same manner as inExample 14. Briefly, ELISA plates were coated with PBS-dilutedanti-human immunoglobulin λ chain antibody (Kirkegaard & PerryLabolatories Inc., 01-10-11) or anti-human immunoglobulin λ chainantibody (Vector, AI-3070) and then serum samples diluted with mouseserum (Sigma, M5905)-supplemented PBS were added. Subsequently,peroxidase-labeled anti-human immunoglobulin μ chain antibody (TheBinding Site Limited, MP008) or peroxidase-labeled anti-humanimmunoglobulin γ chain antibody (Sigma, A0170) was added and incubated.TMBZ (Sumitomo Bakelite, ML-1120T) was added as a peroxidase substrateand then enzyme activity was determined by absorbance measurement at 450nm. Purified human immunoglobulins IgM (Caltag, 13000) and IgG (Sigma,I4506) of known concentrations having μ chain and κ chain were used asstandards for determining human antibody concentrations in the sera bycomparison. These standards were diluted stepwise with mouseserum-supplemented PBS. The results are shown in Table 22. Chimeric miceindividuals were confirmed that had concentrations of complete humanantibody about 40 times higher than in chimeric mice derived from EScells whose endogenous antibody genes were not knocked out. Also,complete human antibody containing human γ chain was confirmed in thesera of the chimeric mice.

[0519] From these results, it was confirmed that the concentration ofcomplete human antibody in which both heavy- and light-chains werederived from human increasesed in the chimeric mice derived from theendogenous antibody heavy-chain and κ chain-deficient mouse ES cellsretaining both a human chromosome #14 partial fragment and a humanchromosome #22 partial fragment. TABLE 22 Concentrations of HumanAntibodies in Chimeric Mice (ELISA) ES Chimeric clone mouse Chimerism(%) IgM, λ (mg/l) IgG, λ (mg/l) LH13 CLH13-1 95 13 2.6 LH13 CLH13-2 902.8 0.36

EXAMPLE 66

[0520] Production of Complete Human Antibody-Producing Hybridomas fromChimeric Mice Prepared by Transferring the Endogenous AntibodyHeavy-Chain and Light-Chain-Deficient Mouse ES Cells Retaining Both aHuman Chromosome #14 Partial Fragment and a Human Chromosome #22 PartialFragment Into Immunodeficient Mouse Host Embryos

[0521] A chimeric mouse CLH13-3 (derived from TT2FES clone LH13;chimerism 35%) obtained in Section 3 of Example 67 was immunized withHSA from day 43 after birth. Briefly, human serum albumin (HSA, Sigma,A3782) dissolved in PBS was mixed with an adjuvant (MPL+TDM Emulsion,RIBI Immunochem Research Inc.) to prepare a HSA solution with aconcentration of 0.25 mg/ml, 0.2 ml of which was administeredintraperitoneally twice at an interval of 1 week. One week thereafter,the mouse was immunized with human serum albumin dissolved in PBS. Themouse was bled every 1 week to determine the concentrations of anti-HSAhuman antibodies in the serum by ELISA in the same manner as in Example14. The results are shown in FIG. 35. The spleen was removed from thechimeric mouse at day 3 after the final immunization and then cellfusion was performed using PEG in the same manner as in Example 24 toprepare hybridomas. Briefly, the fused cells were diluted with a medium(Sanko Pure Chemical, S Cloning Medium CM-B) containing HAT (DainipponPharmaceutical Co., Ltd., No. 16-808-49) or 1 mg/ml of G418 to give aconcentration of 10⁶ spleen cells/ml and then dispensed into 96-wellplates (100 μl/well), followed by cultivation. Both of the selectionmedia contained 5% HCF (Air Brown). At day 6 of the cultivation,colonies were formed in almost all wells in both the G418 selection andHAT selection plates. A total of about 770 hybridoma-positive wells wereobtained. The culture supernatants were collected and subjected toscreening for human antibody-producing hybridomas by ELISA in the samemanner as in Example 14. Briefly, ELISA plates were coated withanti-human immunoglobulin λ chain antibody (Vector, AI-3070).Biotin-labeled anti-human immunoglobulin λ chain antibody (Vector,BA-3070) and an avidin-peroxidase complex (Vector ABC Kit PK4000) wereused for detection with TMBZ (Sumitomo Bakelite, ML-1120T) used as asubstrate. An absorbance about 2 times higher than the absorbance in thenegative control was used as a criterion for judgement. As a result, 17positive wells were obtained. The cells of the positive wells weretransferred into 24-well plates and cultured in IMDM medium containing10% FBS. The culture supernatants were analyzed by ELISA in the samemanner as in Section 4 of Example 65. As a result, the presence of0.09-11 mg/ml of complete human antibody having both human Ig μ & Igλwas confirmed in 16 wells. The antibody titer of anti-HSA human λ chainwas determined in the same manner as in Example 33 to obtain onepositive well. The cells of the well which was complete humanantibody-positive and anti-HSA human λ chain-positive were cloned bylimiting dilution according to the method described in Ando, “MonoclonalAntibody Experiment Procedure Manual”, published by Kodansha Scientificin 1991. As a result, 2 clones of anti-HSA human λ chain-positivehybridomas were obtained.

[0522] From these results, it was confirmed that complete humanantibody-producing hybridomas could be obtained from chimeric miceprepared by transferring the endogenous antibody heavy-chain andlight-chain-deficient mouse ES cells retaining both a human chromosome#14 partial fragment and a human chromosome #22 partial fragment intoimmunodeficient mouse host embryos. Furthermore, it was confirmed thatthe antibody titers of antigen-specific human Igμ and Igλ increased inresponse to the stimulation with the HSA antigen. It was furtherconfirmed that hybridomas producing a HAS-specific antibody consistingof human Igμ and Igλ could be obtained from this chimeric mouse.

[0523] Since the fused cells had a drug resistance marker on theirchromosome, it was possible to select hybridomas using G418 withoutadding HAT. After G418 selection, only those cells having a humanchromosome grow and, thus, hybridomas can be obtained selectively. Also,it is expected that a human chromosome can be prevented from falling offfused cells. Furthermore, it is expected that even myeloma cellsunsuitable for HAT selection such as those having HGPRT(hypoxanthine-guanine-phosphoribosyltransferase) enzyme may becomeavailable for cell fusion.

EXAMPLE 67

[0524] Production of Chimeric Mice with Heavy-Chain Gene-Disrupted HostEmbryos

[0525] From those mice exhibiting agouti coat color among the progeny ofthe endogenous antibody heavy-chain one allele-disrupted TT2F cellclone-derived chimeric mice produced in Example 49, mice retaining thedisrupted allele are selected by Southern blot analysis (Example 49) orthe like (the expected possibility is ½). Offsprings born by the matingof those antibody heavy-chain-deficient heterozygous male and femalemice are subjected to Southern blot analysis (see Example 49), analysisof the production of antibody heavy-chains in sera (Kitamura et al.,Nature, 350:423-, 1991), etc. Thus, antibody heavy-chain-deficienthomozygotes can be obtained which are deficient in both alleles andwhich can hardly produce functional antibodies of their own (theexpected possibility is {fraction (1/4;)} for the results inmembrane-type μ chain-deficient mice, see Kitamura et al., Nature,350:423-, 1991).

[0526] (1) Establishment of an Antibody Heavy-Chain Knockout MouseStrain

[0527] Those mice that exhibited agouti coat color among the progeny ofthe endogenous antibody heavy-chain one allele-disrupted TT2F cellclone-derived chimeric mice produced in Example 49 were subjected toSouthern blot analysis (Example 49) to select those mice that retainedthe disrupted allele. Offsprings born by the mating of these antibodyheavy-chain-deficient heterozygous male and female mice were subjectedto Southern blot analysis (see Example 49) and analysis of theproduction of antibody μ chain in sera (Kitamura et al., Nature, 350:423-, 1991), etc. As a result, antibody heavy-chain-deficienthomozygotes could be obtained which were deficient in both alleles andwhich could hardly produce functional antibodies of their own (for theresults in membrane-type μ chain-deficient mice, see Kitamura et al.,Nature, 350:423-, 1991).

[0528] Thus, an antibody heavy-chain knockout mouse strain could beestablished from the antibody heavy-chain one allele-disrupted TT2F cellclone.

[0529] Embryos obtained by mating the homozygous male and female micebred in a clean environment may be used as hosts for producing chimericmice. In this case, most of the B cells functional in the resultantchimeric mice are derived from the injected ES cells. Other mousestrains which cannot produce their own functional B cells, such asRAG-2-deficient mouse (Sinkai et al., Cell, 68:855-, 1992), may also beused for this purpose. In this system, chimeric mice are produced by thesame procedures as in Example 10, etc. using the mouse ES cell clonefrom Examples 62, 63 or 64 which is deficient in endogenous antibodyheavy-chain & light-chain and which retains human chromosomes #14+#2,#14+#22 or #14+#2+#22 (fragments). The resultant chimeric mice mainlyproduce human antibodies by the expression of human antibody heavy-chain(on chromosome #14), light-chain κ (on chromosome #2) and light-chain λ(on chromosome #22) genes that are functional in ES cell-derived Bcells.

[0530] (2) Detection and Quantitative Determination of Complete HumanAntibody in sera of the Chimeric Mice Produced by Injecting theEndogenous Antibody Heavy-Chain and κ Chain-Deficient Mouse ES CellsRetaining Both a Human Chromosome #2 Partial Fragment and a HumanChromosome #14 Partial Fragment into Immunodeficient Mouse Host Embryos

[0531] Chimeric mice were produced in the same manner as in Section 1 ofExample 65 by injecting the ES cell clone KH10 from Example 62 into theembryos obtained by mating male and female mice of the antibodyheavy-chain knockout mouse strain established in Section 1 of Example67. Seven-week old resultant chimeric mice were bled to determine theconcentrations of human antibodies in the sera by ELISA in the samemanner as in Example 14 and Section 3 of Example 65. The results areshown in Table 23. Complete human antibodies having human μ chain+κchain and human γ chain+κ chain, respectively, were confirmed in thesera of the chimeric mice. It was also confirmed that by transferring EScells into immunodeficient host embryos, complete antibodies could beobtained even in the resultant chimeric mice of low chimerism since Bcells are differentiated only from ES cells. TABLE 23 Concentrations ofHuman Antibodies in Chimeric Mice (ELISA) Chimeric ES clone mouseChimerism (%) IgM, κ (mg/l) IgG, κ (mg/l) KH13 CKH10-1 6 6.1 0.17 KH13CKH10-2 3 1.9 0.4

[0532] (3) Detection and Quantitative Determination of Complete HumanAntibody in sera of the Chimeric Mice Produced by Injecting theEndogenous Antibody Heavy-Chain and κ Chain-Deficient Mouse ES CellsRetaining Both a Human Chromosome #14 Partial Fragment and a HumanChromosome #22 Partial Fragment into Immunodeficient Mouse Host Embryos

[0533] Chimeric mice were produced in the same manner as in Section 2 ofExample 65 by injecting the ES cell clone LH13 from Example 62 into theembryos obtained by mating male and female mice of the antibodyheavy-chain knockout mouse strain established in Section 1 of Example67. Five-week old resultant chimeric mice were bled to determine theconcentrations of human antibodies in the sera by ELISA in the samemanner as in Example 14 and Section 4 of Example 65. The results areshown in Table 24. Complete human antibodies having human μ chain+λchain and human γ chain +λ chain, respectively, were confirmed in thesera of the chimeric mice. TABLE 24 Concentrations of Human Antibodiesin Chimeric Mice (ELISA) Chimeric ES clone mouse Chimerism (%) IgM, λ(mg/l) LH13 CLH13-3 35 51 LH13 CLH13-4 85 32 LH13 CLH13-4 30 27

EXAMPLE 68

[0534] Retention of the Human Chromosome in Offsprings of HumanChromosome #14 Fragment (Containing Antibody Heavy-ChainGene)-Transferred ES Cell-Derived Chimeric Mice

[0535] (1) Isolation of Human-Mouse Hybrid Cells Retaining a HumanChromosome #14 Fragment Containing an Antibody Heavy Chain Gene

[0536] It was observed in Example 42 that a human chromosome #2 fragmenttransferred into mice was transmitted to their progeny. Thus, it isexpected that the possibility of transmission of human chromosome #14 toprogeny will be increased if a fragment of this chromosome is used.A9/#14 clone (Example 9; corresponding to A9/14-C11 clone described inTomizuka et al., Nature Genet. vol 16, 133-143 (1997)) retaining anintact human chromosome #14 marked with a G418 resistance gene wassubjected to a more detailed FISH analysis (Example 9). As a result, itwas observed that about 10% of the cell population contained only a verysmall, fragmented human chromosome #14. This chromosome fragment isalmost of the same size as the chromosome #2 fragment (Example 12) andbelieved to contain the G418 resistance marker.

[0537] In order to isolate the cell clones containing the fragmentedhuman chromosome #14, (about 300) A9/#14 cells were seeded on 10 cmplates and cultured. At day 10 of the cultivation, 31 colonies werepicked up. Genomic DNAs were prepared from these clones and subjected toPCR analysis in the same manner as in Example 9 using chromosome #14specific primers (the 18 primers shown in Example 9 were used except PCIand NP). Out of the 16 primers, only IgM, IGG1, IGA2 and IGVH3 werefound in one clone (A9/SC20). Since D14S543 (Science, HUMAN GENETIC MAP(1994); the base sequence was obtained from databases of GenBank, etc.)which is a marker located near the human chromosome #14 long armtelomere was also detected in the clone, the fragment of interest(hereinafter referred to as “SC20 fragment”) retained in the clone isbelieved to contain a region adjacent to the chromosome #14 telomere andwhich contained an antibody heavy-chain gene.

[0538] SC20 fragment was subjected to FISH analysis (Tomizuka et al.,Nature Genet. vol 16, 133-143 (1997)) using a human chromosome-specificprobe. As a result, it was observed that the size of the chromosome inthe clone that hybridized to the probe was smaller than in the controlclone (containing an intact chromosome #14). Thus, it was confirmed thatA9/SC20 contained a fragment of human chromosome #14.

[0539] Further, in order to examine whether SC20 fragment contained ahuman chromosome #14-derived centromere sequence, chromosome samplesfrom A9/SC20 cells were hybridized to digoxigenin-11-dUTP-labeled humanchromosome #14 or #22-specific α satelite DNA (purchased from COSMOBIO)which was used as a probe, followed by FISH analysis according to themethod described in a reference (Tomizuka et al., Nature Genet. vol 16,133-143 (1997)). As a result, a signal hybridizing to the above probewas confirmed. Thus, it has become clear that SC20 fragment contains ahuman-derived centromere sequence (FIG. 36).

[0540] (2) Transfer of Human Chromosome #14 (Fragment) into TT2F Cellsand Stable Retention of the Chromosome Therein

[0541] A human chromosome #14 fragment was microcell-transferred intoTT2F cells using A9/SC20 as a chromosome donor cell in the same manneras in Example 9. As a result of G418 (300 μg/ml) selection, 5 resistantclones were obtained. These ES cell clones were subjected to PCRanalysis (Example 68, Section 1) and FISH analysis (using humanchromosome-specific probes; Tomizuka et al., supra) to confirm theretention of a human chromosome #14 fragment. The results of the FISHanalysis are shown in FIG. 37.

[0542] Stable retention of transferred human chromosomes in mice isimportant for efficient expression of the transferred genes andefficient transmission of the transferred chromosomes to their progeny.Since selection by addition of drugs is impossible after an ES cellclone has been injected into host embryos, it is desired thattransferred human chromosomes be retained stably even undernon-selective conditions.

[0543] TT2F(SC20)-21 clone containing SC20 fragment was cultured in amedium not containing G418 for a long period to examine the retention ofSC20 fragment under this condition.

[0544] Briefly, TT2F(SC20)-21 clone was cultured in a selective medium(G418: 300 μg/ml) for 1 week and then subcultured in a non-selectivemedium for 45 days (subcultured every other day with 8-fold dilution).At day 0, 15, 30 and 45 of the subculture, 300-1000 cells were seeded insix 35 mm plates, three of which contained the selective medium and theother three non-selective medium. The cells were cultured in theseplates for about 1 week and then the colonies were counted. Chromosomeretention ratio (A/B) was calculated by dividing the total number ofcolonies in the 3 plates under selective conditions (A) by the totalnumber of colonies in the 3 plates under non-selective conditions (B).For the purpose of comparison, an experiment was conducted using P-21clone (Example 40) containing W23 fragment derived from human chromosome#2 in the same manner as described above (the selective medium contained0.75 μg/ml of puromycin). The results are shown in FIG. 38. The valuesshown in FIG. 38 are average values from 3 independent experiments. SC20fragment exhibited a high retention ratio of 95% or above even after the45 day cultivation under non-selective conditions. On the other hand,W23 fragment exhibited a retention ratio of 14% under identicalconditions.

[0545] There have been reports of the transfer of a human Ychromosome-derived artificial chromosome (containing human Y-derivedcentromere) into CHO (hamster fibroblasts), DT40 (chicken B lymphocytes)and mouse ES cell (Shen et al., Hum. Mol. Genet. 6, 1375-1382). Undernon-selective cultivation, the artificial chromosome was retained stablyin CHO and DT40. In mouse ES cells, however, only the chromosome whichaccidentally acquired the mouse centromere as a result of rearrangementwas retained stably. From these results, an opinion was proposed that ahuman-derived centromere is unstable in mouse ES cells (Shen et al.,supra).

[0546] The results described above show that SC20, though it contains ahuman-derived centromere (Example 68, Section 1), is very stable inmouse ES cells. Since the retention of W23 fragment (which is alsosuggested to contain a human-derived centromere) (Tomizuka et al.,supra) in mouse ES cells appeared to be unstable, it is considered thatthe stability of human-derived chromosomes in mouse ES cells variesdepending on the type of the chromosome.

[0547] From these results, it was demonstrated that SC20 fragment isvery useful as a vector for transferring a gene into mice.

[0548] (3) Production of Chimeric Mice from the ES Cell Clone RetainingHuman Chromosome #14 (Fragment)

[0549] Cells in the frozen stock of G418 resistant ES cell cloneTT2F(SC20)-21 which was obtained in Example 68, Section 2 and which wasconfirmed to retain a human chromosome #14 fragment were thawed, startedup for culture and injected into 8-cell stage embryos obtained by matingmale and female mice of ICR (CREA JAPAN, INC.); the injection rate was10-12 cells per embryo. After the embryos were cultured overnight in themedium for ES cells (see Example 9) to develop into blastocysts, about10 of the injected embryos were transplanted to each side of the uterusof foster mother ICR mice (CREA JAPAN, INC.; 2.5 days afterpseudopregnant treatment).

[0550] As a result of transplantation of a total of 188 injectedembryos, 22 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo (ICR)-derived albino coat color (white). Outof the 22 offsprings, 20 mice were recognized to have partial agouticoat color, indicating the contribution of the ES cells. Out of the 20mice, two were chimeric mice in which their coat color was completeagouti (i.e. ES cell-derived).

[0551] From these results, it was confirmed that ES cell cloneTT2F(SC20)-21 retaining a human chromosome #2 fragment maintains theability to produce chimera, that is, the ability to differentiate intonormal tissues of mice.

[0552] Two 5-week old chimeric mice [derived from TT2F(SC20)-21,chimerism 100%, C14m-16 and -17] were bled to determine theconcentrations of human antibody IgM and IgG in the sera by ELISA in thesame manner as in Example 14. The results are shown in Table 25. TABLE25 Concentrations of Human Antibody Heavy-Chains in Chimeric Mice(ELISA) Chimeric mouse Chimerism (%) IgM (mg/l) IgG (mg/l) C14m-16 1007.9 1.0 C14m-17 100 6.0 1.3

[0553] Human antibodies IgM and IgG were detected in the sera of bothchimeric mice. The concentrations of these human antibodies werecomparable to the concentrations in the chimeric mice retaining thelarger human chromosome #14 fragment (see Example 14). Thus, it wasdemonstrated that the human antibody gene contained in SC20 fragment isfunctional.

[0554] (4) Confirmation of the Retention of Human Chromosome in theProgeny of Chimeric Mice Derived from the Mouse ES Cells (TT2F, XO)Retaining a Human Chromosome #14 Fragment, and Detection andQuantitative Determination of Human Antibody μ Chain and γ Chain in seraof the Progeny

[0555] Examination was made as to whether ES cell-derived offspringswould be produced by mating the female chimeric mice C14m-16 and C14m-17(both having 100% chimerism in coat color) from Example 68, Section 3with male ICR mice. By this mating, offsprings with agouti coat colorshould be produced from TT2F cell (agouti: dominant)-derived oocytes inthe chimeric mice fertilized by male ICR mouse (albino:recessive)-derived sperms, and offsprings with albino coat color shouldbe produced from ICR-derived oocytes in the chimeric mice. Actually, allof the viable offspring mice obtained by this mating (30 in total)exhibited ES cell-derived agouti coat color, indicating efficienttransmission of ES cells to the germ cell lineage. Genomic DNAs wereprepared from the tails of these offspring mice to examine the retentionof a human chromosome fragment by PCR. PCR amplification was performedusing the three primers (IGVH3, IgM and D14S543) of which the presencein TT2F(SC20)-21 was confirmed. As a result, the presence of the threemarkers detected in TT2F(SC20)-21 was confirmed in 10 out of the 30offspring mice (33%). These results show that TT2F cell cloneTT2F(SC20)-21 retaining a human chromosome #14 fragment differentiatedinto functional oocytes in the chimeric mice and that the humanchromosome #14 fragment was transmitted to the F₁ progeny derived fromthe oocytes.

[0556] Detection and quantitative determination of human antibodies IgMand IgG in sera were performed on 9 out of the 10 offspring mice whichwere confirmed to retain a human chromosome #14 fragment, as describedbelow. About 4-8 week-old mice were bled to detect human antibody μchain and γ chain by ELISA in the same manner as in Example 14. As aresult, human antibody μ chain and γ chain were detected in the sera ofall of the mice tested (see Table 26). Thus, It was confirmed that thehuman antibody heavy chain gene also functions in the F₁ progeny born bythe chimeric mice. TABLE 26 Concentrations of Human Antibodies IgM andIgG in Chimeric Mice (ELISA) Mother Mouse Chimeric Individual Mouse No.IgM (mg/l) IgG (mg/l) C14m-16 16-5  12.9 2.2 C14m-16 16-14 3.5 2.2C14m-16 16-16 4.1 2.0 C14m-16 16-17 5.5 3.9 C14m-17 17-7  5.7 1.0C14m-17 17-8  3.6 1.2 C14m-17 17-19 3.5 0.75 C14m-17 17-22 2.4 1.4C14m-17 17-23 5.3 1.9

[0557] Further, 3 male mice and 4 female mice in the F₁ progeny weremated with MCH(ICR) mice (purchased from CREA JAPAN, INC.) to obtain F₂progenies, which were subjected to PCR analysis of tail DNA and analysisfor human antibody μ chain expression as described above. As a result,it was confirmed that SC20 fragment was transmitted to 30% of the F₂progeny through F₁ male mice (43 out of the 142 offsprings werepositive) and to 33% of the F₂ progeny through F₁ female mice (20 out ofthe 60 offsprings were positive).

[0558] These results show that a mouse strain was established whichretains the human chromosome #14 fragment (containing a human antibodyheavy chain gene), which expresses human antibody heavy-chains and whichcan transmit the human chromosome to the subsequent generation.

[0559] (5) Stable Retention of a Human Chromosome #14 Fragment in Mice

[0560] Three F₁ mice (16-5, 17-8 and 17-23 shown in Table 26) which wereobtained in Example 68, Section 4 and which retained SC20 fragment wereused in analysis for the ratio of retention of SC20 fragment in mice.The mice were injected intraperitoneally with 0.3 ml of CORCEMID (100μg/ml) and then killed by dislocation of the cervical vertebrae in aneuthanasic manner, followed by removal of the brain, liver, spleen,testis and bone marrow. All of these tissues except the bone marrow werewashed with PBS(−), cut into pieces with scissors for anatomy, givenhypotonic treatment with KCl (0.075 M) for 15 minutes, and fixed inCarnoy's fixative. Specimens were prepared using the supernatant of theCarnoy fixation by conventional methods. FISH analysis was performedusing a human chromosome-specific probe (Human COT-1 DNA) according tothe method described in a reference (Tomizuka et al., Nature Genetics,16, 133-143). As to the brain, spleen, liver and bone marrow, 30 or morenuclei in interphase were selected randomly for each of these tissues.Then, the number of nuclei in which a signal was detected (mark “+” inFIG. 39) and the number of nuclei in which a signal was not detected(mark “−” in FIG. 39) were counted to calculate the retention ratio. Thetestis were classified into the 1st meiosis phase spreads, the 2ndmeiosis phase spreads and sperms. Ten or more spreads or sperms wereselected for each group and then counting was performed in the samemanner as described above to calculate the retention ratio. As a result,all of the 3 mice exhibited a retention ratio of almost 100% in thebrain and liver. A decrease in the retention ratio was observed in thebone marrow and spleen. In the testis, a retention ratio of 80-100% wasobtained for the 1st meiosis phase spreads, and a retention ratio of30-50% for sperms. Assuming that SC20 fragment is retained stably, thetheoretical retention ratio should be 100% for the 1st meiosis phasespreads and 50% for the 2nd meiosis phase spreads and sperms. Thus, itis believed that SC20 fragment is retained stably in the testis.

[0561] At the same time, fibroblasts were prepared from the tail andthen the ratio of retention of SC20 fragment was examined in the samemanner as in Example 79. As a result, the retention ratios in mice 16-5,17-8 and 17-23 were 98%, 96% and 98%, respectively (50 nuclear platewere tested for each mouse).

[0562] (6) Hereditary Relief of Antibody Production Ability-DeficientMice by the Transfer of a Human Chromosome #14 Fragment (ContainingAntibody Heavy-Chain Gene)

[0563] The knockout mouse whose antibody μ chain gene essential for thegeneration of B lymphocytes is disrupted (Section 1, Example 67) cannotproduce antibody because the mouse is deficient in mature B lymphocytesresponsible for humoral immunity. The following experiment was conductedto examine as to whether this deficiency could be relieved bytransferring SC20 fragment (containing a human antibody heavy-chaingene) by mating.

[0564] Those mice exhibiting agouti coat color among the progenyobtained by mating the endogenous antibody heavy-chain oneallele-disrupted TT2F cell clone-derived chimeric mice from Example 49with MCH(ICR) mice were subjected to Southern blot analysis to selectmice retaining the disrupted allele. A female antibodyheavy-chain-deficient heterozygote thus selected was mated with a maleF₁ offspring (17-7) which was obtained in Example 68, Section 4 andwhich retains SC20 fragment. The resultant 5 offspring mice weresubjected to both PCR analysis for confirming the retention of SC20fragment and determination of human antibody μ chain and γ chain in thesera (see Example 68, Section 4). As a result, it was confirmed thatthree mice #2, #3 and #5 retained SC20 fragment (Table 27). Furthermore,as a result of the analysis for mouse antibody μ chain expression(Example 75), it was demonstrated that mice #2 and #3 are mouse μchain-negative, that is, endogenous antibody heavy-chain-deficienthomozygotes (Table 27). These results were consistent with the resultsof Southern blot analysis (see Example 49) using the DNAs prepared fromthe tails of the 5 mice. Compared to mouse #1 in which neither mouse norhuman antibody heavy chain was detected, very high concentrations ofhuman antibody μ chain (310 mg/l) and γ chain (860 mg/l) were detectedin mouse #3 which is antibody heavy-chain-deficient homozygote and whichretains the human chromosome #14 fragment. Further, quantitativedetermination of human γ subclasses was performed on mouse #3 in thesame manner as in Example 29 to detect all of the 4 subclasses (γ1, γ2,γ3 and γ4). In particular, the concentration of human μ chain in thismouse is comparable to the concentration of mouse μ chain in wild-typemice (Mendez et al., Nature Genet. 15, 146-156 (1977)). These resultsshow that the symptom of inability for antibody production because ofdesruption of endogenous heavy-chain gene (deficiency of B lymphocytes:see Kitamura et al., Nature, 350, 423-, 1991) in this mouse was cured bythe transfer of human chromosome #14 fragment SC20 (containing anantibody heavy-chain gene), and that the mouse has recovered the abilityto produce antibody and the ability to produce B lymphocytes. TABLE 27Mouse Retention of Mouse μ Human IgM Human IgG No. SC20 Fragment Chain(mg/l) (mg/l) 1 − − Below detec- 0.33 tion limit 2 + + 8.4 5.3 3 + − 310860 4 − + Not Not measured measured 5 + + 4.8 0.86

EXAMPLE 69

[0565] Retention of the Human Chromosome in Offsprings of HumanChromosome #22 (Fragment)-Transferred ES Cell-Derived Chimeric Mice

[0566] (1) Fragmentation of Human Chromosome #22 using Microcell Fusion

[0567] Since it was observed that both a human chromosome #2 fragment(Example 42) and a human chromosome #14 fragment (Example 68, Section 4)once transferred into mice were transmitted to their offsprings, it isexpected that fragmentation of human chromosome #22 would increase thepossibility of transmission of this chromosome to offspring mice. When ahuman chromosome is transferred into a recipient cell by microcellfusion, it is observed that 40-80% of the transferred clones retain thehuman chromosome which has been fragmented at the time of fusion(Oshimura et al., Protein, Nucleic Acid, Enzyme, vol. 35, No. 14, 1990).The present inventors tried to fragment human chromosome #22 utilizingthis phenomenon.

[0568] A microcell fusion experiment (see Example 1) was conducted usingclone 6-1 from Example 35 as a chromosome donor cell and wild-type mouseA9 cells as a recipient cell, thereby producing seventy-three G418resistant clones. Genomic DNAs were prepared from the resultant clonesand then screened by PCR using Ig A primers (Example 2). Sixty-sevenclones which retained human IgA gene were subjected to PCR analysisusing primers specific to 8 markers located on human chromosome #22(D22S315, D22S275, D22S280, D22S278, D22S283, D22S272, D22S282 andD22S274; for the order of location on human chromosome #22, see Nature,vol. 377, 367-379 (1995); base sequences for these primers were obtainedfrom databases of such as GenBank). As a result, it was found that apart of the markers disappeared in 25 clones. Thus, it was suggestedthat chromosome #22 was fragmented in these clones (FIG. 40). Amongthem, clone #22 and clone #28 are considered to have a fairly smallfragment, because markers other than Ig λ and D22S315 disappeared in theformer and markers other than Igλ disappeared in the latter (FIG. 40).Clone #28 was subjected to FISH analysis (see Example 18) using a humanchromosome-specific probe. The results are shown in FIG. 41. It isobserved that the size of the chromosome hybridizing to the probe issmaller in this clone than in the control clone (containing an intactchromosome #22). Thus, a human chromosome #22 fragment containingantibody λ gene could be obtained as a result of fragmentation whichoccurred at the time of microcell fusion.

[0569] (2) Transfer of Chromosome #22 (Fragment) into TT2F Cells

[0570] Chromosome #22 (fragment) was microcell-transferred into TT2Fcells by the method described in Example 2, using clones #22, #28 and6-1 as chromosome donor cells. Clones #22 and A9/#22(6-1) were subjectedto puromycin (0.75 μg/ml) selection, and clone #28 was subjected to G418(225 μg/ml) selection. As a result, drug resistant clones were obtainedas follows: 13 from clone #22, 5 from clone 6-1 and 3 from clone #28.These ES cell clones are subjected to PCR analysis and FISH analysis toconfirm the retention of human chromosome #22 (fragment) in the samemanner as in Example 69, Section 1.

[0571] (3) Production of Chimeric Mice from ES Cell Clones RetainingHuman Chromosome #22 (Fragment)

[0572] In the same manner as in Example 3, chimeric mice are producedfrom the drug resistant ES cell clones which were obtained in Example69, Section 2 and which were confirmed to retain human chromosome #22.Confirmation of the retention of human chromosome #22 (fragment) in theresultant chimeric mice is performed by the method described in Example

[0573] (4) Transmission of Human Chromosome #22 (Fragment) to Offsprings

[0574] The chimeric mice retaining human chromosome #22 (fragment) aremixed and mated with ICR mice. Retention of a human chromosome #22fragment in the offsprings is examined by PCR using genomic DNAsprepared from the tails of the offspring mice having agouti coat color(see Examples 30, 42 and 43). As shown in Examples 42 and 43, mouse EScell clones retaining human chromosome #22 or a fragment thereof candifferentiate into oocytes or sperms functional in chimeric mice,thereby allowing to the human chromosome #22 (fragment) to betransmitted their progenies. Thus, it is possible to establish a mousestrain which retains human chromosome #22 (fragment) containing humanantibody light-chainλ gene and which can transmit it to the subsequentgeneration.

EXAMPLE 70

[0575] Production of Mice Retaining Both Human Chromosome #2 (Fragment)and #14 (Fragment) by Mating

[0576] The human chromosome #2 (fragment)-retaining mouse strain fromExample 42 or 43 is mated with the human chromosome #14(fragment)-retaining mouse strain from Example 68 to produce offsprings.Genomic DNAs are prepared from the tails of the offspring mice. The DNAis analyzed by PCR, etc. (Examples 9, 42 and 43) to produce those micewhich retain both human chromosome #2 partial fragment and humanchromosome #14 (fragment).

EXAMPLE 71

[0577] Production of Mice Retaining Both Human Chromosome #22 (Fragment)and #14 (Fragment) by Mating

[0578] The human chromosome #22 (fragment)-retaining mouse strain fromExample 69 is mated with the human chromosome #14 (fragment)-retainingmouse strain from Example 68 to produce offsprings. Genomic DNAs areprepared from the tails of the offspring mice. The DNA is analyzed byPCR, etc. (Examples 30, 42 and 43) to produce those mice which retainboth human chromosome #22 (fragment) and #14 (fragment).

EXAMPLE 72

[0579] Production of Mice Retaining the Three Human Chromosomes #2(Fragment), #14 (Fragment) and #22 (Fragment) by Mating

[0580] The mouse strain retaining both human chromosome #2 (fragment)and #14 (fragment) obtained in Example 71 is mated with the mouse strainretaining a human chromosome #2 fragment obtained in Example 42 or 43 toproduce offsprings. Genomic DNAs are prepared from the tails of theoffspring mice. The DNA is analyzed by PCR, etc. (Examples 9, 30, 42 and43) to produce those mice which retain all of the three humanchromosomes, #22 (fragment), #14 (fragment) and #2 (fragment).Alternatively, mice retaining all of the above three human chromosomesmay also be obtained by mating the mouse strain retaining both humanchromosome #2 (fragment) and #14 (fragment) from Example 70, with themouse strain retaining a human chromosome #22 fragment from Example 69.

EXAMPLE 73

[0581] Production of a Complete Human Antibody-Producing Mouse Strain byMating

[0582] The mouse strains retaining human chromosomes #2+#14 (Example70), #14+#22 (Example 71) and #2+#14+#22 (Example 72), respectively, arerepeatedly mated with a mouse strain deficient in endogenous antibodyheavy-chain and light-chain κ genes. From the resultant offsprings,those mouse strains which retain human chromosomes #2+#14, #14+#22 or#2+#14+#22 and which are homozygotes in the deficiency of endogenousantibody heavy-chain and light-chain κ genes, are selected by PCRanalysis, etc. (Examples 9, 30, 42 and 43). In these strains, completehuman antibodies are mainly produced (Green et al., Nature Genetics, 7:13-, 1994; Lonberg et al., Nature, 368:856-, 1994).

[0583] Hereinbelow, the establishment of a mouse strain which retainsboth a human chromosome #2 fragment and a human chromosome #14 fragmentand which is homozygote in the deficiency of endogenous antibodyheavy-chain and light-chain κ genes will be described. The 4 strainsused for the mating and the method for assaying the genotypes of eachstrain are as follows.

[0584] (1) The mouse strain from Example 42 retaining a human chromosome#2 fragment: the retention of the human chromosome #2 fragment isassayed by PCR analysis of the tail-derived DNA as described in Example42 and by the expression of human antibody κ chain in the sera.

[0585] (2) The mouse strain from Example 68, Section 4 retaining a humanchromosome #14 fragment: the retention of the human chromosome #14fragment is assayed by PCR analysis of the tail-derived DNA as describedin Example 68, Section 4 and by the expression of human antibody μ chainin the sera.

[0586] (3) The antibody heavy-chain knockout mouse strain from Example67, Section 1: heavy-chain deficiency-homozygotes or heterozygotes wereassayed by Southern blot analysis of the tail-derived DNA as describedin Example 67, Section 1 and by the presence or absence of theexpression of mouse antibody μ chain in the sera (see Example 75).

[0587] (4) The antibody κ chain knockout mouse strain from Example 80: κchain deficiency-homozygotes or heterozygotes were assayed by Southernblot analysis of the tail-derived DNA as described in Example 80.

[0588] A mouse strain which retains all of the 4 genotypes (i.e.,retaining a human chromosome #2 fragment, retaining a human chromosome#14 fragment, antibody heavy-chain-deficiency homozygote orheterozygote, and antibody κ chain-deficiency homozygote orheterozygote) was established by mating the above 4 strains with eachother. Specifically, after the above 4 strains used as startingmaterials were mated several times, a male mouse having the genotypes of“retaining the human chromosome #14 fragment, antibodyheavy-chain-deficiency homozygote and antibody κ chain-deficiencyheterozygote” was mated with a female mouse having the genotypes of“retaining the human chromosome #2 fragment, antibodyheavy-chain-deficiency homozygote and antibody κ chain-deficiencyhomozygote” or “retaining the human chromosome #2 fragment, antibodyheavy-chain-deficiency homozygote and antibody κ chain-deficiencyheterozygote” or “retaining the human chromosome #2 fragment, antibodyheavy-chain-deficiency heterozygote and antibody κ chain-deficiencyhomozygote”. As a result, mouse HK23 “retaining the human chromosome #2fragment, retaining the human chromosome #14 fragment, antibodyheavy-chain-deficiency homozygote and antibody κ chain-deficiencyheterozygote” and mouse HK29 “retaining the human chromosome #2fragment, retaining the human chromosome #14 fragment, antibodyheavy-chain-deficiency heterozygote or wild-type, and antibody κchain-deficiency heterozygote” were obtained. FIG. 42 shows theconcentration of each antibody in the sera and the genotypes of thesemice, together with the data on mouse HK28 which was also produced bythe above-described mating and which has the genotypes of “retaining thehuman chromosome #2 fragment, retaining the human chromosome #14fragment, antibody heavy-chain-deficiency heterozygote or wild-type, andantibody κ chain-deficiency wild-type”. A complete human antibodyconsisting of human μ chain and human κ chain was detected at aconcentration of 18 mg/l in the serum of mouse HK23 (Example 38).

[0589] It is possible to produce those mice having the genotypes of“retaining the human chromosome #2 fragment, retaining the humanchromosome #14 fragment, antibody heavy-chain-deficiency homozygote andantibody κ chain-deficiency homozygote” by mating the mice obtained bythe above mating with each other. In this mouse strain, it is expectedthat human antibody κ chain will be expressed at a higher concentrationthan in mouse HK23 because the deficiency of the endogenous κ chain geneis substituted by the human antibody κ chain gene contained in the humanchromosome #2 fragment (Lonberg et al., Nature, 368, 856-, 1994). It isalso expected that the concentration of a complete human antibodyconsisting of human heavy-chain and human κ chain will increase further.

EXAMPLE 74

[0590] Production of a Human Antibody-Producing Hybridoma From a MouseStrain which is Obtained by Mating and Which Retains a HumanChromosome(s) Containing a Human Antibody Gene(s)

[0591] The mice retaining a human chromosome(s) containing a humanantibody gene(s) which were obtained in Example 42, 43, 68, 69, 70, 71,72 or 73 are immunized with an antigen of interest in the same manner asin Example 25. The spleen is removed from each mice and the spleen cellsare fused with myeloma cells to produce hybridomas. After cultivationfor 1-3 weeks, the culture supernatant is analyzed by ELISA. The ELISAis performed by the method described in Examples 14, 15, 21, 22, 25, 33,34, 37 and 38. As a result, human antibody positive clones and cloneswhich are human antibody positive and specific to the antigen used inthe immunization are obtained.

EXAMPLE 75

[0592] Detection and Determination of Mouse IgM in sera of Chimeric MiceDerived from the Mouse Antibody Heavy-Chain Both Alleles-Disrupted TT2FCell Clone

[0593] Offspring mice were born in the same manner as in Example 40 fromthe mouse antibody heavy-chain both alleles-disrupted TT2F cell clone(#131-3) from Example 51. Three mice having chimerisms of 0%, 50% and99%, respectively, were selected from the offspring mice. Mouse IgM intheir sera was detected and determined. Briefly, the chimeric mice ofabout 2 weeks after birth were bled and mouse IgM concentration in thesera was determined by ELIZA by the same procedures as in Example 14. APBS-diluted anti-mouse IgM antibody (Kirkegaard & Perry LaboratoriesInc., 01-18-03) was fixed, and then a PBS-diluted serum samplesupplemented with 5% FBS was added. Peroxidase-labeled anti-mouse IgMantibody (Kirkegaard & Perry Laboratories Inc., 074-1803) was added andthe absorbance at 450 nm was determined using TMBZ as a substrate.Purified mouse IgM (Pharmingen, 0308ID) was used as a standard. Thisstandard was diluted stepwise with FBS-supplemented PBS. The results areshown in Table 28. Of the chimeric mice derived from the mouse antibodyheavy-chain both alleles-disrupted TT2F cells, the mouse having achimerism of 99% exhibited a low mouse IgM concentration. Thus, it wasconfirmed that the mouse heavy-chain gene from the ES cells hardlyfunctions in this mouse. TABLE 28 Concentration of Mouse IgM in ChimericMice (ELISA) Chimerism % IgM (mg/l) 0 12 50 11 99 1.5

EXAMPLE 76

[0594] Preparation of a Targeting Vector for Knocking Out AntibodyLight-Chain κ gene in ES Cells

[0595] Plasmid LoxP-PGKPuro in which LoxP sequence was inserted at bothends of a puromycin resistance gene was prepared in the same manner asin Example 48, Section 1. Briefly, a puromycin resistance cassettePGKPuro was cut out from PGKPuro plasmid DNA (Watanabe et al., Biochem.Biophys. Res. Commun. 213:130-137 (1995); released from Peter W. Laird,Whitehead Institute for Biochemical Research and Massachusetts Instituteof Technology, Dept. of Biology, Cambridge, Mass.) using restrictionenzyme SailI and then blunted. PGKPuro was inserted into the SmaI andEcoRV restriction sites of a LoxP-sequence containing plasmid to produceplasmid pLoxP-PGKPuro (FIG. 30). Further, a DNA fragment comprising agenomic DNA constant region containing the mouse antibody light-chainκ Jregion and constant region was replaced with the LoxP-PGKPuro gene inthe same manner as in Example 48 (FIG. 31).

EXAMPLE 77

[0596] Production of an Antibody Light-Chain Gene Both Alleles-DisruptedStrain Antibody Light-Chain-Deficient-Heterozygote (and AntibodyHeavy-Chain-Deficient-Homozygote) Mouse ES Cells

[0597] (1) A Puromycin Resistance Gene was Inserted into the AntibodyLight-Chain Deficient-Heterozygote TT2F Clone (HD43) Obtained in Example58 to Give a Strain in which Both Alleles of a Light-Chain Gene wereDisrupted.

[0598] The antibody light-chain targeting vector prepared in Example 76was linearized with restriction enzyme KpnI to transform HD43 clone inthe same manner as in Example 58. The resultant transformants weresubjected to selective culture at a puromycin concentration of 0.75μg/ml. At day 7-9 of the cultivation, colonies formed were picked up. Apart of these colonies was stored frozen, and the remaining part wasused to prepare genomic DNA in the same manner as in Example 49. GenomicDNAs from the puromycin resistant strains were digested with restrictionenzyme EcoRI (Takara Shuzo), separated by agarose gel electrophoresisand subjected to Southern blot analysis to detect homologousrecombinants using the probe described in Example 48, Section 4 (seeExamples 58 and 59). As a result, 4 clones in which both alleles of anantibody light-chain were disrupted were obtained from the 74 clonesanalyzed. Under usual culture conditions, no changes in growth rate andmorphology were observed in these clones, as compared to the TT2F clonebefore gene disruption. This suggests that the clones underconsideration retain the ability to produce chimera.

[0599] (2) Production of Chimeric Mice from the AntibodyHeavy-Chain-Deficient-Homozygote and Antibody Light-Chain Gene BothAlleles-Disrupted Clone

[0600] Cells in the frozen stock of antibody light-chain gene bothalleles-disrupted TT2F cell clone HD43P-10 from Example 77, Section 1were thawed, started up for culture and injected into 8-cell stageembryos obtained by mating male and female mice of ICR (CREA JAPAN,INC.); the injection rate was 10-12 cells per embryo. After the embryoswere cultured overnight in the medium for ES cells (see Example 9) todevelop into blastocysts, about 10 of the injected embryos weretransplanted to each side of the uterus of foster mother ICR mice (CREAJAPAN, INC.; 2.5 days after pseudopregnant treatment).

[0601] As a result of transplantation of a total of 161 injectedembryos, 37 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo (ICR)-derived albino coat color (white). Outof the 37 offsprings, 9 mice were recognized to have partial agouti coatcolor, indicating the contribution of the ES cells. Out of the 9 mice,four were chimeric mice in which more than 80% of their coat color wasagouti (i.e. ES cell-derived).

[0602] From these results, it was confirmed that antibody light-chainboth alleles-disrupted ES cell clone HD43P-10 maintains a high abilityto produce chimera.

EXAMPLE 78

[0603] Removal of the G418 Resistance and Puromycin Resistance MarkerGenes from the Antibody Light-Chain Deficient-Homozygote (and AntibodyHeavy-Chain Deficient-Homozygote) TT2F Cell Clone

[0604] From the antibody light-chain both alleles-disrupted HD43P-10clone (puromycin resistant, G418 resistant) which was obtained andconfirmed to have a high chimera-forming ability in Example 77, thepuromycin resistance and G418 resistance marker genes were removed bythe procedures described in Example 52. Briefly, an expression vectorpBS185 (BRL) containing a Cre recombinase gene which causes asite-specific recombination between the LoxP sequences inserted at bothends of the G418 resistance marker gene was transferred into the clonedescribed above in the same manner as in Example 52. The resultantpuromycin (0.75 μg/ml) sensitive clones (6 clones) were grown toconfluence in 35 mm plates in the same manner as in Example 52. Threefifths (⅗) of the resultant culture were suspended in 0.5 ml of apreservation medium [ES medium+10% DMSO (Sigma)] and stored frozen at−80° C. The remaining two fifth (⅖) were divided into two portions andinoculated into two 12-well gelatin-coated plates. Cells in one platewere cultured in non-selective medium for 2 days. Cells in other platewere cultured in the presence of 300 μg/ml of G418 for 2 days. As aresult, 5 puromycin sensitive and G418 sensitive clones which would bekilled in the presence of G418 were obtained.

EXAMPLE 79

[0605] Increase in the Expression of Human Antibody κ Chain in sera as aResult of Mating a Human Chromosome #2 Fragment-Retaining Mouse Strainwith C57BL/6 Strain

[0606] The hereditary background of the progenies of the chimeric mice[hereinafter referred to as “F₁(chimera×MCH)”] which were described inExamples 43 and 44 and which retain a human chromosome #2 fragment(hereinafter referred to as “W23 fragment”) is that they are a mixtureof TT2F cell (Example 39)-derived CBA mouse strain and C57BL/6 mousestrain, and MCH(ICR) mouse strain mated with the chimeric mice. In orderto observe the behavior of W23 fragment under a hereditary background ashomogeneous as possible, first, F₁(chimera×MCH) were back-crossed withMCH(ICR). The offspring mice obtained by the mating of F₁(chimera×MCH)(randomly selected 8 male and 6 female mice)×MCH(ICR) were examined asto whether they would retain W23 fragment in the same manner as inExample 43. As a result, it was confirmed that W23 fragment wastransmitted through male to 8% of the offsprings (25 out of the 324offsprings were positive) and through female to 22% of the offsprings(32 out of the 148 offsprings were positive). When the resultantF₂(F₁×MCH)(randomly selected 8 male and 8 female mice) were furthermated with MCH(ICR), the transmission ratio was 9% through male (30 outof the 346 offsprings were positive) and 24% through female (48 out ofthe 202 offsprings were positive). Thus, the results was similar to thatobtained by the mating of F₁(chimera×MCH)×MCH(ICR). F₃(F₂×MCH) wereobtained by the latter mating.

[0607] The concentrations of human antibody κ chain in the sera of4-12-week old chimeric mice (FIG. 43, indicated as “Chimera”, 4 mice),F₁(chimera×MCH) (19 mice), F₂(F₁×MCH) (39 mice) and F₃(F₂×MCH) (33 mice)were determined in the same manner as in Example 44. The results areshown in FIG. 43. Human antibody κ chain was detected in all of the miceretaining W23 fragment. On the other hand, the κ chain concentrationsvaried greatly in F₂(F₁×MCH) and F₃(F₂×MCH); the averaged values inthese groups were lower than those in the chimeric mice andF₁(chimera×MCH).

[0608] In order to examine the influence which would be caused by themating with a strain other than MCH(ICR), the same F₂(F₁×MCH) mice asused in the experiment of mating with MCH(ICR) were mated with C57BL/6N(purchased from CREA JAPAN, INC.). Concentrations of κ chain weredetermined in the same manner on the resultant 26 mice retaining W23fragment [F₃(F₂×C57BL/6)]. As a result, these mice exhibited κ chainconcentrations as high as those in the chimeric mice and F₁(chimera×MCH)(FIG. 43). As described above, F₃(F₂×MCH) and F₃(F₂×C57BL/6) are derivedfrom the same F₂(F₁×MCH) mice as one of the parents. Therefore, it isbelieved that the difference between F₃(F₂×MCH) and F₃(F₂×C57BL/6) is intheir hereditary background alone. Thus, it is indicated that differencein hereditary background influences the amount of expression of humanantibody chain. Further, it has become clear that the hereditarybackground of C57BL/6 is more desirable than that of MCH(ICR) forefficient expression of human antibody κ chain. From similarexperiments, it has been demonstrated that the hereditary background ofC3H HeN (purchased from CREA JAPAN, INC.) is comparable to or betterthan that of C57BL/6 for efficient expression of human antibody κ chain.

[0609] The following experiment was conducted to examine as to whetherthe influence of hereditary background on antibody κ chainconcentrations observed above is related to the ratio of chromosomeretention (stability) at the level of individual mice. Briefly,metaphase chromosome samples were prepared from tail-derived fibroblastsand bone marrow cells of 2F-1 mouse (serum κ chain concentration: 84mg/l) and 1F-3 mouse (serum κ chain concentration: 13 mg/l) inF₁(chimera×MCH) and subjected to FISH analysis (Tomizuka et al., NatureGenetics, vol 16, 133-143). The ratio of those metaphase spreadscontaining W23 fragment hybridizing to a human chromosome-specific probeto all of the spreads observed was determined. It is believed that theresultant values represent the W23 fragment retention ratios infibroblasts and bone marrow cells, respectively. As a result, withrespect to 2F-1, the retention ratio was 51% in fibroblasts and 34% inbone marrow cells; and with respect to 1F-3, the retention ratio was 23%in fibroblasts and 18% in bone marrow cells (more than 50 nuclear platewere measured for each case). These results suggest that the κ chainconcentrations in sera correlated to the ratios of retentions of W23fragment in fibroblasts and bone marrow cells. In other words, it isvery likely that hereditary background influences the stability of thetransferred human chromosome fragment itself. Thus, it is believed thatthe hereditary background of C57BL/6 or C3H strain is desirable forefficient expression of a gene not only on the chromosome #2 fragmentdescribed herein but also on other chromosome fragments (e.g. chromosome#14 fragment).

[0610] In order to verify the above conjecture, a male mouse #17-7 inF₁(chimera×MCH) which retains the human chromosome #14 fragment obtainedin Example 68 (hereinafter referred to as “SC20 fragment”) and whichexpresses a human antibody heavy-chain in the serum was mated withMCH(ICR) and C57BL/6. Of the resultant offsprings, two F₂(F₁×MCH) miceand two F₂(F₁×C57BL/6) mice, each retaining SC20 fragment, weresubjected to determination of human antibody heavy-chain concentrationsin the sera (see Example 68). Furthermore, metaphase chromosome sampleswere prepared from the tails of these mice and then the ratio of SC20fragment retention was determined in the same manner as for W23fragment. As a result, the human μ chain concentration was 11.0 mg/l and1.1 mg/l and the chromosome retention ratio 74% and 54% in F₂(F₁×MCH),whereas the human μ chain concentration was 47 mg/l and 54 mg/l and thechromosome retention ratio 84% and 88% in F₂(F₁×C57BL/6). F₂(F₁×C57BL/6)mice exhibited higher values in both the human μ chain concentration andthe chromosome retention ratio. Thus, it has become clear that thehereditary background of C57BL/6 is desirable for stable retention of atransferred human chromosome and for efficient expression of a genelocated thereon, as presumed from the results obtained on W23fragment-retaining mice.

EXAMPLE 80

[0611] Production of Chimeric Mice from an AntibodyHeavy-Chain-Deficient and Antibody κ Chain-Homologous Recombinant ESCell Clone

[0612] Cells in the frozen stock of antibody heavy-chain-deficient andantibody κ chain-homologous recombinant ES cell clone HD43 from Example58 were thawed, started up for culture and injected into 8-cell stageembryos obtained by mating male and female mice of ICR (CREA JAPAN,INC.); the injection rate was 10-12 cells per embryo. After the embryoswere cultured overnight in the medium for ES cells (see Example 9) todevelop into blastocysts, about 10 of the injected embryos weretransplanted to each side of the uterus of foster mother ICR mice (CREAJAPAN, INC.; 2.5 days after pseudopregnant treatment).

[0613] As a result of transplantation of a total of 314 injectedembryos, 51 offspring mice were born. Chimerism in the offsprings can bedetermined by the extent of TT2 cell-derived agouti coat color (darkbrown) in the host embryo (ICR)-derived albino coat color (white). Outof the 51 offsprings, 26 mice were recognized to have partial agouticoat color, indicating the contribution of the ES cells. Out of the 26mice, two were chimeric female mice in which 100% of their coat colorwas agouti (i.e. ES cell-derived).

[0614] From these results, it was confirmed that antibodyheavy-chain-deficient and antibody light-chain-homologous recombinant EScell clone HD43 maintains the ability to produce chimera. In the femalemice exhibiting 100% contribution, it is highly possible that the EScells have been differentiated into functional germ cells (oocytes).

[0615] Examination was made as to whether ES cell-derived offspringswould be produced by mating the above female chimeric mice (both having100% chimerism in coat color) with male ICR mice. By this mating,offsprings with agouti coat color should be produced from TT2F cell(agouti: dominant)-derived oocytes in the chimeric mice fertilized bymale ICR mouse (albino: recessive)-derived sperms, and offsprings withalbino coat color should be produced from ICR-derived oocytes. Actually,all of the viable offspring mice obtained by one mating for each femalemouse exhibited ES cell-derived agouti coat color. Genomic DNAs wereprepared from the tails of these offspring mice to examine the presenceof an antibody κ chain disrupted allele by Southern blot analysis(Example 58). As a result, mice having an antibody κ chain disruptedallele were obtained.

[0616] Twenty-seven offspring mice produced by the mating of theantibody light-chain deficient-heterozygote male and female mice weresubjected to Southern blot analysis (Example 58). As a result, antibodylight-chain wild-type alleles disappeared and only disrupted alleleswere observed in 7 offspring mice. Hence, these mice were believed to beantibody light-chain-deficient homozygotes. FIG. 44 shows the results ofdetection and quantitative determination of mouse antibody κ chain and λchain in the sera.

[0617] In those mice which were judged to be antibodylight-chain-deficient homozygotes by the Southern blot analysis (Nos. 4,6, 14, 22, 24, 25 and 26 in FIG. 44), the concentrations of κ chain aregreatly reduced (the remaining κ chain appears to be derived from theirmother mice). Instead, the concentrations of λ chain are greatlyincreased in these mice. These results are consistent with the reportedresults of analysis of the antibody κ chain knockout mouse (Yong-Rui Zouet al., EMBO J. 12, 811-820 (1993)).

[0618] Thus, an antibody κ chain knockout mouse strain could beestablished from antibody κ chain homologous recombinant ES cell cloneHD43.

EXAMPLE 81

[0619] Preparation of a Targeting Vector for Inserting Human TelomereSequence into Human Chromosome #22

[0620] Fragmentation of human chromosome #22 on which human antibody λchain gene (hereinafter referred to as “Igλ gene”) was located wasattempted by inserting human telomere sequence by homologousrecombination (J. E. Itzhaki et al., Nature Genet., 2, 283-287, 1992).Specifically, a targeting vector for inserting human telomere sequenceinto the LIF locus located very close to Ig λ gene (on the telomereside) was prepared.

[0621] Human telomere sequence was synthesized by PCR according to themethod of J. J. Harrington et al. (Nature Genet., 15, 345-355, 1997).The PCR product was purified by agarose gel electrophoresis and thenblunted with DNA Blunting Kit (Takara Shuzo). The blunted PCR productwas inserted into the Eco RV site of pBluescript SK II(+) (Toyobo) byligation using DNA Ligation Kit (Takara Shuzo) (pBS-TEL). This plasmidpBS-TEL was sequenced. As a result, it was found that the telomeresequence had been inserted in the following direction: HindIII-(TTAGGG)n-Eco RI.

[0622] Subsequently, the LIF gene region on human chromosome #22 to beused in the homologous recombination was amplified by PCR as describedbelow, and then cloned into plasmid PBS-TEL. The sequences of theprimers used in the PCR were as follows. Sense primer:5′-TCGAACTAGTAGGAGAAGTGAACTTGAGGAGGC3′ (SEQ ID NO: 65) Antisense primer:5′-TCGAACTAGTGATTCAGTGATGCTGTGCAGG-3′ (SEQ ID NO: 66)

[0623] The PCR reaction mixture was composed of 5 μl of 10×LA PCR bufferII (Mg²⁺ free) (Takara Shuzo); 5 μl of 25 mM MgCl₂; 8 μl of dNTP mixture(2.5 mM each) (Takara Shuzo); 10 pmol of sense primer; 10 pmol ofantisense primer; 100 ng of template DNA (HFL1, genomic DNA from primaryculture human fibroblasts); 0.5 μl of LA Taq (5 U/μl) (Takara Shuzo) andsterile distilled water to make a total volume of 50 μl. All of theoperations for preparing the reaction mixture were carried out on ice.Then, reaction tubes were placed in the well of a thermal cycler (PCRSystem 9600, Perkin-Elmer) preset at 85° C. After the tubes were heatedat 94° C. for 1 minute, 35 cycles of reaction were carried out at 98 °C. for 10 seconds and at 65° C. for 5 minutes. The PCR product waspurified and then digested with Spe I (Spe I site was present in theprimers), followed by insertion into the Spe I site in pBS-TEL. Theplasmid in which the LIF gene had been inserted in a opposite directionof the human telomere sequence (TTAGGG)n was selected (M. Giovannini etal., Cytogenet Cell Genet 64, 240-244, 1993) (pBS-TEL/LIF).

[0624] Subsequently, plasmid pGKpuro containing a puromycin resistancegene (S. Watanabe et al., Biochem. Biophys. Res. Comm., 213, 130-137,1995) was digested with Eco RI and blunted, followed by insertion of NotI linker. The puromycin resistance gene was cut out by digesting theresultant plasmid with Not I and then inserted into the Not I site ofpBS-TEL/LIF. The plasmid in which the direction of transcription of thepuromycin resistance gene was the same as that of the LIF gene wasselected (pBS-TEL/LIFPuro, see FIG. 45). The resultant plasmid wasamplified in E. coli DH5, purified with QUIAGEN column (Funakoshi) andused for transfection (as described later).

EXAMPLE 82

[0625] Transfer of Human Chromosome #22 into Chicken DT40 Cells

[0626] Mouse A9 cells containing human chromosome #22 marked with a G418resistance gene (Tomizuka et al., Nature Genet., vol 16, 133-143, 1997;hereinafter referred to as “A9/#22neo”) were cultured in Dulbecco'smodified Eagle's Minimal Essential Medium (hereinafter referred to asDMEM”) supplemented with 10% fetal bovine serum and G418 (800 μg/ml).Chicken DT40 cells were cultured in DMEM supplemented with 10% FBS, 1%chicken serum and 10⁻⁴ M 2-mercaptoethanol.

[0627] Microcells were prepared as described below (for details, seeShimizu et al., “Cell Technology Handbook”, published by Yodosha, p.127-). A9/#22neo cells were cultured in twelve 25 cm² centrifuge flasks(Costar) until the cell concentration reached about 90% saturation.Then, the medium was exchanged with a medium (DMEM+20% FBS) supplementedwith COLCEMID (0.07 μg/ml; demecolcine, Wako Pure Chemical Industries,Inc.). The cells were cultured for another 2.5-3 days to formmicrocells. Thereafter, the culture solution was removed from thecentrifuge flasks, into which a solution of cytochalasin B (10 μg/ml,Sigma) prewarmed at 37° C. was filled and centrifuged at 34° C. at 8000rpm for 1 hour. The microcells were suspended in DMEM and purified byfiltration with filters. After the purification, the microcells werecentrifuged at 1500 rpm for 10 minutes and then suspended in 5 ml ofDMEM. DT40 cells ( 2×10⁷) were centrifuged at 1000 rpm for 5 minutes,washed with DMEM twice and suspended in 5 ml of DMEM. The microcellsprepared above were re-centrifuged at 1500 rpm for 10 minutes and then,without removal of the supernatant, 5 ml of the previously prepared DT40suspension was overlayered gently. After centrifugation at 1300 rpm for5 minutes, the supernatant was removed. The cell pellet was suspended in2 ml of PHA-P (100 μg/ml, DIFCO) and left to stand in an incubator at37° C. under 5% Co₂ for 15 minutes. Then, the suspension was centrifugedat 1700 rpm for 7 minutes. The supernatant was removed and the cellpellet was loosened by tapping. To the cell pellet, 1 ml of PEG1500(polyethylene glycol, Boehringer) was added gently and the pellet wastreated for 1.5-2 minutes under agitation. After this treatment, 1 ml ofDMEM was added over approximately 1 minute. Then, 3 ml of DMEM was addedover approximately 2 minutes. Thereafter, DMEM was added to make a totalvolume of 11 ml and the resultant mixture was mixed gently. The mixturewas left to stand for 10 minutes at room temperature and thencentrifuged at 1300 rpm for 5 minutes. The supernatant was removed. Thecells were suspended in 10 ml of the above-described culture medium andcultured in φ 100 mm plates for 24 hours. Twenty-four hours later, themedium was exchanged with one supplemented with G418 (1 mg/ml). Theresultant culture was dispensed into three 24-well plates (SumitomoBakelite), followed by selective culture for about 2 weeks to isolateG418 resistant clones.

[0628] (1) PCR Analysis

[0629] As a result of the selective culture, about thirty G418 resistantclones were obtained. Genomic DNAs were extracted from these clonesusing Puregene DNA Isolation Kit (Gentra System). Using the genomic DNAas a template, PCR was performed with human Ig λ gene-specific primersto identify clones having human chromosome #22 containing Igλ gene. TheIgλ gene-specific primers used were as follows.5′-GAGAGTTGCAGAAGGGGTGACT-3′ (SEQ ID NO: 67)5′-GGAGACCACCAAACCCTCCAAA-3′ (SEQ ID NO: 68)

[0630] The PCR reaction mixture was composed of 5 μl of 10×Ex Taq buffer(Takara Shuzo); 8 μl of dNTP mixture (2.5 mM each) (Takara Shuzo); 10pmol of each primer; 100 ng of genomic DNA; 0.5 μl of Ex Taq (5 U/μl )(Takara Shuzo) and sterile distilled water to make a total volume of 50μl. All of the operations for preparing the reaction mixture werecarried out on ice. Then, reaction tubes were placed in the well of athermal cycler (PCR System 9600, Perkin-Elmer) preset at 85° C. Afterthe tubes were heated at 94° C. for 1 minute, 35 cycles of reaction werecarried out at 98° C. for 10 seconds, at 56° C. for 30 seconds and at72° C. for 30 seconds. As a result, 2 clones having human Ig λ gene wereidentified. The presence of polymorphic markers (D22S315, D22S280,D22S283 and D22S274; Polymorphic STS Primer Pair, BIOS; J. E. Collins etal., Nature 377 suppl.: 367, 1995) located on human chromosome #22 weredetected in these clones by PCR (FIG. 46). The PCR conditions were thesame as used for the detection of human Ig λ gene. Mark “◯” indicatesthat the marker was detected. Mark “X “indicates that the marker was notdetected. The diagram at the left side shows the location of each markeron chromosome #22 based on a physical map. From these results, it wassuggested that these 2 clones have a almost intact human chromosome #22.As to the other clones, although human Ig λ gene was not detected, someof the polymorphic markers on chromosome #22 described above weredetected.

[0631] (2) FISH Analysis

[0632] One of the above 2 clones (clone No. 52-18) was subjected to FISHanalysis to examine how the human chromosome #22 actually existed incells. Basic operations such as preparation of chromosome samples,hybridization and detection were performed according to Tomizuka et al.(Nature Genet. 16, 133-143, 1997). As a probe, human COT-1 DNA (labeledwith Rhodamine) was used. As a result of observation of 20-30 spreads,it was confirmed that an almost intact human chromosome #22 was presentindependently (FIG. 50). Those stained in red are human chromosome #22.

[0633] From these results of analysis, it was thought that chicken DT40cell clone 52-18 (hereinafter referred to as “DT40/#22neo”) has intacthuman chromosome #22.

EXAMPLE 83

[0634] Targeted Truncation of Human Chromosome #22 in Chicken DT40 Cells

[0635] DT40/#22neo from Example 82 was transfected with plasmidpBS-TEL/LIFPuro prepared in Example 81 and an attempt was made toperform targeted truncation of the human chromosome #22 on the LIFlocus.

[0636] DT4O//#22neo cells were cultured under the same conditions asdescribed in Example 82 in the presence of G418 (1 mg/ml). 10⁷ cellswere washed with cold PPS once, suspended in 0.5 ml of PBS and placed onice. Then, 25-30 μg of pBS-TEL/LIFPuro linearized with Eco RI was addedto the cells, mixed with a pipette, transferred into an electroporationcuvette (Bio-Rad) and left to stand in ice for 10 minutes. The cuvettewas set in a gene pulser (Bio-Rad) and then a voltage of 550 V wasapplied at a capacitance of 25 μF. After the cuvette was left to standon ice for 10 minutes, the cells were transferred into 72 cm² cultureflasks containing the above-described medium and cultured for 24 hours.Twenty-four hours later, the medium was exchanged with a mediumsupplemented with G418 (1 mg/ml) and puromycin (0.3 μg/ml, Sigma). Theresultant culture was dispensed into five to eight 96-well cultureplates, followed by selective culture for about 2 weeks to isolateresistant clones.

[0637] (1) PCR Analysis

[0638] As a result of the selective culture, about 80 resistant cloneswere obtained. Genomic DNAs were extracted from these cells as describedabove and subjected to PCR to identify homologous recombinants in whicha human telomere sequence was integrated into the LIF locus. One of theprimers was designed such that its sequence was complementary to a partof the LIF gene region which was not contained in the vector (see FIG.47). The other primer was designed such that its sequence wascomplementary to a part of the puromycin resistance gene which wascontained in the vector. The sequences of the primers are as follows.Puro.1: 5′-GAGCTGCAAGAACTCTTCCTCACG-3′ (SEQ ID NO: 69) LIF1:5′-ATGACTCTAAGGCAGGAACATCTGTACC-3′ (SEQ ID NO: 70)

[0639] The PCR reaction mixture was composed of 5 μl of 10×LA PCR bufferII (Mg²⁺ free) (Takara Shuzo); 5 μl of 25 mM MgCl₂; 8 μl of dNTP mixture(2.5 mM each) (Takara Shuzo); 10 pmol of each primer; 100 ng of templateDNA; 0.5 μl of LA Taq (5 U/μl ) (Takara Shuzo) and sterile distilledwater to make a total volume of 50 μl. All of the operations forpreparing the reaction mixture were carried out on ice. Then, reactiontubes were placed in the well of a thermal cycler (PCR System 9600,Perkin-Elmer) pre-set at 85° C. After the tubes were heated at 94° C.for 1 minute, 35 cycles of reaction were carried out at 98° C. for 10seconds and at 65° C. for 10 minutes. A 6.3 kb PCR product as shown inFIG. 47 should be detected only in the homologous recombinants ofinterest. As a result of the PCR, this 6.3 kb band was detected in 8clones (homologous recombination ratio: about 10%). When this PCRproduct was digested with Sal I, a cut pattern was obtained in exactlythe same way as expected. Thus, it was confirmed that these 8 cloneswere homologous recombinants.

[0640] Subsequently, whether or not the truncation occurred as expectedin these 8 clones was examined by PCR detection of the presence of genes(Igλ, LIF, MB, IL2RB, CYP2D6, DIA1, ECGF1 and ARSA; J. E. Collins etal., Nature 377 suppl:367, 1995) and polymorphic markers (D22S315,D22S275, D22S280, D22S281, D22S277, D22S278, D22S283, D22S272, D22S282and D22S274; J. E. Collins et al., Nature 377 suppl.:367, 1995) onchromosome #22.

[0641] A part of the primer sequences used is as described below. Theremaining primer sequences were the same as used by Tomizuka et al.(Nature Genet. 16, 133-143, 1997). The presence of LIF is evident fromthe experiment described above. CYP2D6 Sense primer:5′-CTGCGTGTGTAATCGTGTCC-3′ (SEQ ID NO:71) Antisense primer:5′-TCTGCTGTGAGTGAACCTGC-3′ (SEQ ID NO:72) ECGF1 Sense primer:5′-AGGAGGCACCTTGGATAAGC-3′ (SEQ ID NO:73) Antisense primer:5′-TCACTCTGACCCACGATACAGC-3′ (SEQ ID NO:74)

[0642] The PCR reaction mixture was composed of 5 μl of 10×Ex Taq buffer(Takara Shuzo); 8 μl of dNTP mixture (2.5 mM each) (Takara Shuzo); 10pmol of each primer; 100 ng of genomic DNA; 0.5 μl of Ex Taq (5 U/μl )(Takara Shuzo) and sterile distilled water to make a total volume of50μl. All of the operations for preparing the reaction mixture werecarried out on ice. Then, reaction tubes were placed in the well of athermal cycler (PCR System 9600, Perkin-Elmer) pre-set at 85° C. Afterthe tubes were heated at 94° C. for 1 minute, 35 cycles of reaction werecarried out at 98 ° C. for 10 seconds, at 56° C. (65° C. for CYP2D6 andECGF1) for 30 seconds and at 72° C. for 30 seconds. The results areshown in FIG. 48. Marks “◯” and “X ” have the same meanings as describedabove. As is clear from this Figure, none of the genes and markerslocated on the telomere side of the LIF locus into which a humantelomere sequence had been integrated were detected in clones 67, 68,328 and 343. It is suggested that truncation by the integration of atelomere sequence did occur as expected in at least those 4 clones.

[0643] (2) FISH Analysis

[0644] Whether the human chromosome #22 had been actually truncated ornot was examined by FISH analysis. The experimental method was the sameas described above. As probes, human COT1 DNA (labeled with Rhodamine)and plasmid pGKPuro (labeled with FITC) were used. By COT1 staining, thehuman chromosome #22 can be visually checked for truncation incomparison with DT40/#22neo having intact human chromosome #22.Furthermore, if the chromosome #22 is truncated as expected on the LIFlocus into which the vector has been integrated, a signal from the Puroprobe should be detected at one end of the telomere of the chromosome#22 fragment. A part of the results is shown in FIG. 49. As a result ofobservation of 20-30 spreads for each clone, a small fragment of humanchromosome #22 (red) having a Puro probe-derived signal (yellow green)at one end of the telomere was surprisingly observed in all of the 8homologous recombinant clones. As for clones 64, 212, 222 and 305 whichwere presumed not to have undergone truncation from the results of thePCR analysis, cells having intact chromosome #22 occupied about 10% ofall cells.

[0645] These experimental results show that homologous recombinants inwhich a human telomere sequence has been integrated into the LIF locuscan be obtained at an efficiency of about 10% in chicken DT40/#22neocells and that truncation of the human chromosome #22 has occurred atthe integration site in all of the homologous recombinants (efficiency100%).

1 74 1 20 DNA Artificial Sequence Description of Artificial SequencePrimer 1 tggaaggtgg ataacgccct 20 2 22 DNA Artificial SequenceDescription of Artificial Sequence Primer 2 tcattctcct ccaacattag ca 223 20 DNA Artificial Sequence Description of Artificial Sequence Primer 3gcaatcggtc tgccggaaga 20 4 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 4 ttggatcact ttggacccag 20 5 20 DNAArtificial Sequence Description of Artificial Sequence Primer 5ctctcctgca gggccagtca 20 6 22 DNA Artificial Sequence Description ofArtificial Sequence Primer 6 tgctgatggt gagagtgaac tc 22 7 20 DNAArtificial Sequence Description of Artificial Sequence Primer 7agtcagggca ttagcagtgc 20 8 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 8 gctgctgatg gtgagagtga 20 9 20 DNAArtificial Sequence Description of Artificial Sequence Primer 9tggtggctga aagctaagaa 20 10 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 10 ccagaagaat ggtgtcatta 20 11 20 DNAArtificial Sequence Description of Artificial Sequence Primer 11tccaggttct gcagagcaag 20 12 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 12 tgtagttgga ggccatgtcc 20 13 20 DNAArtificial Sequence Description of Artificial Sequence Primer 13ccccacccat gatccagtac 20 14 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 14 gccctcagaa gacgaagcag 20 15 22 DNAArtificial Sequence Description of Artificial Sequence Primer 15gagagttgca gaaggggtga ct 22 16 22 DNA Artificial Sequence Description ofArtificial Sequence Primer 16 ggagaccacc aaaccctcca aa 22 17 20 DNAArtificial Sequence Description of Artificial Sequence Primer 17ggctatgggg acctgggctg 20 18 22 DNA Artificial Sequence Description ofArtificial Sequence Primer 18 cagagacaca ggcacgtaga ag 22 19 20 DNAArtificial Sequence Description of Artificial Sequence Primer 19ttaagggtca cccagagact 20 20 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 20 tgtagttgga ggccatgtcc 20 21 20 DNAArtificial Sequence Description of Artificial Sequence Primer 21caaaaagtcc aaccctatca 20 22 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 22 gccctcagaa gacgaagcag 20 23 20 DNAArtificial Sequence Description of Artificial Sequence Primer 23tcgttcctgt cgaggatgaa 20 24 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 24 tcactccgaa gctgcctttc 20 25 21 DNAArtificial Sequence Description of Artificial Sequence Primer 25atgtacagga tgcaactcct g 21 26 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 26 tcatctgtaa atccagcagt 20 27 20 DNAArtificial Sequence Description of Artificial Sequence Primer 27gatcccatcg cagctaccgc 20 28 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 28 ttcgccgagt agtcgcacgg 20 29 22 DNAArtificial Sequence Description of Artificial Sequence Primer 29gatgaactag tccaggtgag tt 22 30 22 DNA Artificial Sequence Description ofArtificial Sequence Primer 30 ccttttggct tctactcctt ca 22 31 20 DNAArtificial Sequence Description of Artificial Sequence Primer 31atagagggta cccactctgg 20 32 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 32 aaccaggtag gttgatatgg 20 33 20 DNAArtificial Sequence Description of Artificial Sequence Primer 33aagttcctgt gatgtcaagc 20 34 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 34 tcatgagcag attaaacccg 20 35 20 DNAArtificial Sequence Description of Artificial Sequence Primer 35tgtgaaggag gaccaggtgt 20 36 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 36 tgtaggggtt gacagtgaca 20 37 20 DNAArtificial Sequence Description of Artificial Sequence Primer 37ctgagagatg cctctggtgc 20 38 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 38 ggcggttagt ggggtcttca 20 39 20 DNAArtificial Sequence Description of Artificial Sequence Primer 39ggtgtcgtgg aactcaggcg 20 40 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 40 ctggtgcagg acggtgagga 20 41 20 DNAArtificial Sequence Description of Artificial Sequence Primer 41gcatcctgac cgtgtccgaa 20 42 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 42 gggtcagtag caggtgccag 20 43 20 DNAArtificial Sequence Description of Artificial Sequence Primer 43agtgagataa gcagtggatg 20 44 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 44 gttgtgctac tcccatcact 20 45 21 DNAArtificial Sequence Description of Artificial Sequence Primer 45ttgtatttcc aggagaaagt g 21 46 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 46 ggagacgagg gggaaaaggg 20 47 27 DNAArtificial Sequence Description of Artificial Sequence Primer 47atggactgga cctggaggrt cytctkc 27 48 27 DNA Artificial SequenceDescription of Artificial Sequence Primer 48 atggagyttg ggctgasctggstttyt 27 49 27 DNA Artificial Sequence Description of ArtificialSequence Primer 49 atgrammwac tktgkwbcwy sctyctg 27 50 20 DNA ArtificialSequence Description of Artificial Sequence Primer 50 cagaggcagttccagatttc 20 51 20 DNA Artificial Sequence Description of ArtificialSequence Primer 51 tgggatagaa gttattcagc 20 52 27 DNA ArtificialSequence Description of Artificial Sequence Primer 52 atggacatgrrrdycchvgy kcasctt 27 53 28 DNA Artificial Sequence Description ofArtificial Sequence Primer 53 ccaagcttca ggagaaagtg atggagtc 28 54 28DNA Artificial Sequence Description of Artificial Sequence Primer 54ccaagcttag gcagccaacg gccacgct 28 55 28 DNA Artificial SequenceDescription of Artificial Sequence Primer 55 ccaagcttca gaggcagttccagatttc 28 56 28 DNA Artificial Sequence Description of ArtificialSequence Primer 56 gggaattcgg gtagaagtca ctgatcag 28 57 28 DNAArtificial Sequence Description of Artificial Sequence Primer 57gggaattcgg gtagaagtca cttatgag 28 58 28 DNA Artificial SequenceDescription of Artificial Sequence Primer 58 gggaattcgg gtagaagtcacttacgag 28 59 60 DNA Artificial Sequence Description of ArtificialSequence Synthetic probe 59 accttcatcg tcctcttcct cctgagcctc ttctacagcaccaccgtcac cctgttcaag 60 60 60 DNA Artificial Sequence Description ofArtificial Sequence Synthetic probe 60 tgatgctgca ccaactgtat ccatcttcccaccatccagt gagcagttaa catctggagg 60 61 22 DNA Artificial SequenceDescription of Artificial Sequence Primer 61 ctggggtgag ccggatgttt tg 2262 22 DNA Artificial Sequence Description of Artificial Sequence Primer62 ccaacccagc tcagcccagt tc 22 63 36 DNA Artificial Sequence Descriptionof Artificial Sequence Synthetic oligonucleotide 63 aattcccgcgggtcgacgga tccctcgagg gtacca 36 64 36 DNA Artificial SequenceDescription of Artificial Sequence Synthetic oligonucleotide 64gggcgcccag ctgcctaggg agctcccatg gttcga 36 65 33 DNA Artificial SequenceDescription of Artificial Sequence Primer 65 tcgaactagt aggagaagtgaacttgagga ggc 33 66 31 DNA Artificial Sequence Description ofArtificial Sequence Primer 66 tcgaactagt gattcagtga tgctgtgcag g 31 6722 DNA Artificial Sequence Description of Artificial Sequence Primer 67gagagttgca gaaggggtga ct 22 68 22 DNA Artificial Sequence Description ofArtificial Sequence Primer 68 ggagaccacc aaaccctcca aa 22 69 24 DNAArtificial Sequence Description of Artificial Sequence Primer 69gagctgcaag aactcttcct cacg 24 70 28 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 70 atgactctaa ggcaggaaca tctgtacc 28 71 20DNA Artificial Sequence Description of Artificial Sequence Primer 71ctgcgtgtgt aatcgtgtcc 20 72 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 72 tctgctgtga gtgaacctgc 20 73 20 DNAArtificial Sequence Description of Artificial Sequence Primer 73aggaggcacc ttggataagc 20 74 22 DNA Artificial Sequence Description ofArtificial Sequence Primer 74 tcactctgac ccacgataca gc 22

What is claimed is:
 1. A method for producing a chimeric non-humananimal, which comprises preparing a microcell containing a foreignchromosome(s) or a fragment(s) thereof and transferring the foreignchromosome(s) or fragment(s) into a pluripotent cell by fusion with themicrocell.
 2. A method for producing a pluripotent cell containing aforeign chromosome(s) or a fragment(s) thereof, which comprisespreparing a microcell containing a foreign chromosome(s) or afragment(s) thereof and transferring the foreign chromosome(s) orfragment(s) thereof into a pluripotent cell by fusion with themicrocell.
 3. The method of claim 1 or 2, wherein the foreignchromosome(s) or fragment(s) thereof is(are) larger than 670 kb.
 4. Themethod of claim 3, wherein the foreign chromosome(s) or fragment(s)thereof is(are) at least 1 Mb in length.
 5. The method of claim 1 or 2,wherein the foreign chromosome or fragment thereof contains a regionencoding an antibody.
 6. The method of claim 1 or 2, wherein themicrocell containing a foreign chromosome(s) or a fragment(s) thereof isinduced from a hybrid cell prepared by the fusion of a cell from whichthe foreign chromosome(s) or fragment(s) thereof is(are) derived, with acell having a high ability to form a microcell.
 7. The method of claim6, wherein the microcell containing a foreign chromosome(s) or afragment(s) thereof is induced from a cell prepared by a further fusionof the microcell induced from said hybrid cell with a cell having a highability to form a microcell.
 8. The method of claim 6, wherein the cellfrom which the foreign chromosome(s) or fragment(s) thereof is(are)derived is a human normal diploid cell.
 9. The method of any one ofclaims 6-8, wherein the cell having a high ability to form a microcellis a mouse A9 cell.
 10. The method of claim 1 or 2, wherein thepluripotent cell is one selected from embryonal carcinoma cells,embryonic stem cells, embryonic germ cells and mutants thereof.
 11. Themethod of claim 1 or 2, wherein the foreign chromosome or fragmentthereof contains a gene of interest and the pluripotent cell has adisrupted gene identical with or homologous to said gene of interest onthe foreign chromosome or fragment thereof.
 12. The method of claim 11,wherein the foreign chromosome or fragment thereof contains at least twogenes of interest and the pluripotent cell has disrupted genes identicalwith or homologous to said genes of interest on the foreign chromosomeor fragment thereof.
 13. The method of claim 11, wherein one or bothalleles of a gene identical with or homologous to the gene of intereston the foreign chromosome or fragment thereof are disrupted in thepluripotent cell.
 14. The method of claim 11, wherein the gene ofinterest is an antibody gene.
 15. The method of claim 14, wherein theantibody gene is one or more sets of antibody heavy-chain andlight-chain genes.
 16. The method of claim 1, wherein the foreignchromosome or fragment thereof contains a gene of interest and saidforeign chromosome or fragment thereof is transferred into a pluripotentcell having a disrupted gene identical with or homologous to said geneof interest and then, a chimera is produced from the pluripotent cell byusing an embryo of a non-human animal in a strain deficient in anendogenous gene identical with or homologous to said gene of interest.17. The method of claim 16, wherein the non-human animal in a straindeficient in an endogenous gene identical with or homologous to the geneof interest is produced by homologous recombination in gene targeting.18. The method of claim 1, wherein the chimeric non-human animal retainsthe foreign chromosome(s) or fragment(s) thereof, expresses the gene(s)on the foreign chromosome(s) or fragment(s) thereof, and can transmitthe foreign chromosome(s) or fragment(s) thereof to its progeny.
 19. Themethod of claim 1, wherein the chimeric non-human animal is a mammal.20. The method of claim 19, wherein the mammal is a mouse.
 21. Apluripotent cell containing a foreign chromosome(s) or a fragment(s)thereof.
 22. The cell of claim 21, wherein the foreign chromosome(s) orfragment(s) thereof is(are) larger than 670 kb.
 23. The cell of claim21, wherein the foreign chromosome or fragment thereof contains a geneof interest and the pluripotent cell has a disrupted endogenous geneidentical with or homologous to said gene of interest on the foreignchromosome or a fragment thereof.
 24. The cell of claim 23, wherein theforeign chromosome or fragment thereof contains at least two genes ofinterest and the pluripotent cell has disrupted endogenous genesidentical with or homologous to said genes of interest on the foreignchromosome or a fragment thereof.
 25. The cell of claim 23, wherein oneor both alleles of an endogenous gene identical with or homologous tothe gene of interest are disrupted in the pluripotent cell.
 26. The cellof claim 21, wherein the foreign chromosome or fragment thereof containsan antibody gene.
 27. The cell of claim 26, wherein the antibody gene isone or more sets of antibody heavy-chain and light-chain genes.
 28. Thecell of claim 21, wherein the pluripotent cell is one selected fromembryonal carcinoma cells, embryonic stem cells, embryonic germ cellsand mutants thereof.
 29. A chimeric non-human animal retaining a foreignchromosome(s) or a fragment(s) thereof and expressing a gene(s) on theforeign chromosome(s) or fragment(s) thereof, or its progeny retainingthe foreign chromosome(s) or fragment(s) thereof and expressing thegene(s) on the foreign chromosome(s) or fragment(s) thereof.
 30. Thechimeric non-human animal or its progeny of claim 29, wherein theforeign chromosome(s) or fragment(s) thereof is(are) larger than 670 kb.31. The chimeric non-human animal or its progeny of claim 29, whereinthe foreign chromosome or fragment thereof contains a gene of interestand the animal has a disrupted endogenous gene identical with orhomologous to said gene of interest.
 32. The chimeric non-human animalor its progeny of claim 31, wherein the foreign chromosome or fragmentthereof contains at least two genes of interest and said animal hasdisrupted endogenous genes identical with or homologous to said genes ofinterest.
 33. The chimeric non-human animal or its progeny of claim 31,wherein one or both alleles of an endogenous gene identical with orhomologous to said gene of interest are disrupted.
 34. The chimericnon-human animal or its progeny of claim 31, wherein the gene ofinterest is an antibody gene.
 35. The chimeric non-human animal or itsprogeny of claim 34, wherein the antibody gene is one or more sets ofantibody heavy-chain and light-chain genes.
 36. A non-human animal whichcan be produced by mating the chimeric non-human animals or theirprogenies of claim 29, said non-human animal retaining the foreignchromosome(s) or fragment(s) thereof and expressing the gene(s) on theforeign chromosome(s) or fragment(s) thereof, or its progeny retainingthe foreign chromosome(s) or fragment(s) thereof and expressing thegene(s) on the foreign chromosome(s) or fragment(s) thereof.
 37. Anon-human animal retaining the foreign chromosome(s) or fragment(s)thereof and expressing a gene(s) on the foreign chromosome(s) orfragment(s) thereof, which can be produced by mating the chimericnon-human animal or its progeny of claim 29, or the non-human animal orits progeny of claim 36, with a non-human animal in a strain deficientin said gene(s) or a gene homologous thereto, or its progeny retainingthe foreign chromosome(s) or fragment(s) thereof and expressing thegene(s) on the foreign chromosome(s) or fragment(s) thereof.
 38. Atissue from the chimeric non-human animal or its progeny of claim 29 orfrom the non-human animal or its progeny of claim 36 or from thenon-human animal or its progeny of claim
 37. 39. A cell from thechimeric non-human animal or its progeny of claim 29 or from thenon-human animal or its progeny of claim 36 or from the non-human animalor its progeny of claim
 37. 40. The cell of claim 39, which is a B cell.41. A hybridoma prepared by the fusion of the cell of claim 40 with amyeloma cell.
 42. A method for producing a biologically activesubstance, which comprises expressing the gene(s) on the foreignchromosome(s) or fragment(s) thereof in the chimeric non-human animal orits progeny of claim 29, the non-human animal or its progeny of claim 36or the non-human animal or its progeny of claim 37, or a tissue or acell thereof, and recovering the biologically active substance as anexpression product.
 43. The method of claim 42, wherein the cell of thechimeric non-human animal is a B cell.
 44. The method of claim 43,wherein the B cell is immortalized by fusion with a myeloma cell. 45.The method of claim 42, wherein the biologically active substance is anantibody.
 46. The method of claim 45, wherein the antibody is anantibody of a mammal.
 47. The method of claim 46, wherein the antibodyof a mammal is a human antibody.
 48. A biologically active substancewhich can be produced by the method of claim
 42. 49. A non-human animalretaining at least one human antibody gene larger than 670 kb andexpressing the gene.
 50. The non-human animal of claim 49, which retainsat least one human antibody gene of at least 1 Mb and expresses thegene.
 51. The non-human animal of claim 49, wherein the human antibodygene is selected from the group consisting of human heavy-chain gene,human light-chain κ gene, human light-chain λ gene, and combinationsthereof.
 52. The non-human animal of claim 49, which is deficient in anon-human animal antibody gene identical with or homologous to the humanantibody gene.
 53. The non-human animal of claim 52, wherein thedeficiency of said non-human animal antibody gene is caused bydisrupting the non-human animal antibody gene by homologousrecombination.
 54. A hybridoma prepared by the fusion of a spleen cellof the non-human animal of claim 49 with a myeloma cell.
 55. An antibodyproduced by the hybridoma of claim
 54. 56. A non-human animal expressingat least one class or subclass of human antibody.
 57. The non-humananimal of claim 56, which is deficient in an endogenous antibody geneidentical with or homologous to the expressed class or subclass of humanantibody gene.
 58. The non-human animal of claim 56, wherein the classor subclass of human antibody is selected from IgM, IgG, IgE, IgA, IgDand their subclasses, and combinations thereof.
 59. A non-human animalretaining a foreign DNA(s) larger than 670 kb and expressing a gene(s)on the foreign DNA(s).
 60. The non-human animal of claim 59, which isdeficient in an endogenous gene identical with or homologous to theexpressed gene on the foreign DNA.
 61. The non-human animal of claim 59which retains a foreign DNA(s) of at least 1 Mb and expresses thegene(s) on the foreign DNA(s).
 62. The non-human animal of claim 61,which is deficient in an endogenous gene identical with or homologous tothe expressed gene on the foreign DNA.
 63. A method for producing atransgenic non-human animal, which comprises preparing a microcellcontaining a foreign chromosome(s) or a fragment(s) thereof,transferring the foreign chromosome(s) or fragment(s) into a culturedcell derived from a blastcyst by fusion with the microcell andtransplanting the nucleus of the cultured cell into an enucleatedunfertilized egg.
 64. A pluripotent cell in which at least twoendogenous genes are disrupted.
 65. The cell of claim 64, in which eachof the endogenous genes is disrupted in one or both alleles.
 66. Thecell of claim 64, wherein the disrupted endogenous genes are antibodygenes.
 67. The cell of claim 66, wherein the antibody genes are antibodyheavy-chain and light-chain genes.
 68. The cell of claim 64, wherein thepluripotent cell is one selected from embryonal carcinoma cells,embryonic stem cells, embryonic germ cells and mutants thereof.
 69. Amethod of producing the cell of claim 64 by at least two homologousrecombinations.
 70. The method of claim 69, which comprises the stepsof: disrupting one allele of the endogenous gene in the pluripotent cellby homologous recombination using a drug-resistant marker gene;culturing the pluripotent cell in the presence of the drug to selectdrug-resistant cells; and screening the selected drug-resistant cells toyield a cell in which both alleles of the endogenous gene have beendisrupted.
 71. The method of claim 69, in which one allele of theendogenous gene in the pluripotent cell is disrupted by homologousrecombination using a drug-resistant marker gene and the other allele ofthe endogenous gene is disrupted by another homologous recombinationusing a drug-resistant marker gene.
 72. The method of claim 71, whereinthe same drug-resistant marker gene is used in the two homologousrecombinations.
 73. The method of claim 71, wherein differentdrug-resistant marker genes are used in the two homologousrecombinations.